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Purification, properties and primary structure of alanine dehydrogenase involved in taurine metabolism in the anaerobe Bilophila wadsworthia
Abstract
Alanine dehydrogenase [l-alanine:NAD+ oxidoreductase (deaminating), EC 1.4.1.4.] catalyses the reversible oxidative deamination of l-alanine to pyruvate and, in the anaerobic bacterium Bilophila wadsworthia RZATAU, it is involved in the degradation of taurine (2-aminoethanesulfonate). The enzyme regenerates the amino-group acceptor pyruvate, which is consumed during the transamination of taurine and liberates ammonia, which is one of the degradation end products. Alanine dehydrogenase seems to be induced during growth with taurine. The enzyme was purified about 24-fold to apparent homogeneity in a three-step purification. SDS-PAGE revealed a single protein band with a molecular mass of 42 kDa. The apparent molecular mass of the native enzyme was 273 kDa, as determined by gel filtration chromatography, suggesting a homo-hexameric structure. The N-terminal amino acid sequence was determined. The pH optimum was pH 9.0 for reductive amination of pyruvate and pH 9.0–11.5 for oxidative deamination of alanine. The apparent K
m values for alanine, NAD+, pyruvate, ammonia and NADH were 1.6, 0.15, 1.1, 31 and 0.04 mM, respectively. The alanine dehydrogenase gene was sequenced. The deduced amino acid sequence corresponded to a size of 39.9 kDa and was very similar to that of the alanine dehydrogenase from Bacillus subtilis.
... The sources of the chemicals and gases (N 2 /CO 2 ) used were given elsewhere [22]. Sulfoacetaldehyde was synthesized as bisulfite adduct [23] and its identity confirmed by NMR and IR (K. ...
... The initial step (I) is a pyruvate-dependent transamination of taurine to sulfoacetaldehyde and alanine. Oxidative deamination of alanine to pyruvate and ammonia is catalysed by alanine dehydrogenase [22]. Desulfonation of sulfoacetaldehyde in a presumably thiamine pyrophosphate-dependent reaction (III) yields acetate and putatively sulfite. ...
... Step 1 was modified for purification of larger amounts of Tpa. Crude extract was applied to a DEAE-Sepharose anionexchange column (80 mL) [22] instead of the Mono Q column. Tpa eluted at about 0.1 m NaCl. ...
Bilophila wadsworthia RZATAU is a Gram-negative bacterium which converts the sulfonate taurine (2-aminoethanesulfonate) to ammonia, acetate and sulfide in an anaerobic respiration. Taurine:pyruvate aminotransferase (Tpa) catalyses the initial metabolic reaction yielding alanine and sulfoacetaldehyde. We purified Tpa 72-fold to apparent homogeneity with an overall yield of 89%. The purified enzyme did not require addition of pyridoxal 5'-phosphate, but highly active enzyme was only obtained by addition of pyridoxal 5'-phosphate to all buffers during purification. SDS/PAGE revealed a single protein band with a molecular mass of 51 kDa. The apparent molecular mass of the native enzyme was 197 kDa as determined by gel filtration, which indicates a homotetrameric structure. The kinetic constants for taurine were: Km = 7.1 mm, Vmax = 1.20 nmol·s-1, and for pyruvate: Km = 0.82 mm, Vmax = 0.17 nmol·s-1. The purified enzyme was able to transaminate hypotaurine (2-aminosulfinate), taurine, beta-alanine and with low activity cysteine and 3-aminopropanesulfonate. In addition to pyruvate, 2-ketobutyrate and oxaloacetate were utilized as amino group acceptors. We have sequenced the encoding gene (tpa). It encoded a 50-kDa peptide, which revealed 33% identity to diaminopelargonate aminotransferase from Bacillus subtilis.
... [8]), although there is no theoretical objection to the reactions in Scheme 1 occurring in anaerobes. Indeed, precisely this pathway was found or suspected in several anaerobic micro-organisms [9], and the transaminase and the dehydrogenase have been purified from the strictly anaerobic, taurine-reducing bacterium Bilophila wadsworthia [10,11]. We have now been able to purify the sulpho-lyase from the strictly anaerobic, taurine-fermenting bacterium Desulfonispora thiosulfatigenes GKNTAU [12,13] and we describe its properties and primary sequence. ...
... Natural organosulphonates are widespread in oxic and anoxic ecosystems [9,32,33], and their role, especially that of taurine, in the nutrition of anaerobic mats has recently been detailed [32]. Sulphoacetaldehyde is the point of convergence of several degradative pathways [9], so the enzymes and genes described here and elsewhere [10,11,27] should be the start of an understanding of aspects of these ecosystems at the molecular level. ...
The strictly anaerobic bacterium Desulfonispora thiosulfatigenes ferments taurine via sulphoacetaldehyde, which is hydrolysed to acetate and sulphite by sulphoacetaldehyde sulpho-lyase (EC 4.4.1.12). The lyase was expressed at high levels and a two-step, 4.5-fold purification yielded an apparently homogeneous soluble protein, which was presumably a homodimer in its native form; the molecular mass of the subunit was about 61 kDa (by SDS/PAGE). The mass was determined to be 63.8 kDa by matrix-assisted laser-desorption ionization-time-of-flight (MALDI-TOF) MS. The purified enzyme converted 1 mol of sulphoacetaldehyde to 1 mol each of sulphite and acetate, but no requirement for thiamine pyrophosphate (TPP) was detected. The N-terminal and two internal amino acid sequences were determined, which allowed us to generate PCR primers. The gene was amplified and sequenced. The DNA sequence had no significant homologue in the databases searched, whereas the derived amino acid sequence indicated an oxo-acid lyase, revealed a TPP-binding site and gave a derived molecular mass of 63.8 kDa.
... The reverse reaction was assayed by following the disappearance of sulfoacetaldehyde after derivatization (Denger et al., 2001) in a reaction mixture with the same buffer with PLP but with 2 mmol sulfoacetaldehyde and 10 mmol alanine. Alanine dehydrogenase (Ald) was routinely measured photometrically as the reduction of NAD (Laue & Cook, 2000b) during oxidative deamination. Occasionally the reaction was confirmed by the assay of reductive amination of pyruvate following alanine formation or NADH oxidation (Laue & Cook, 2000b). ...
... Alanine dehydrogenase (Ald) was routinely measured photometrically as the reduction of NAD (Laue & Cook, 2000b) during oxidative deamination. Occasionally the reaction was confirmed by the assay of reductive amination of pyruvate following alanine formation or NADH oxidation (Laue & Cook, 2000b). Sulfoacetaldehyde acetyltransferase (Xsc) was assayed by GC as the thiamin diphosphate (ThDP)-and phosphate-dependent release of acetate after acidification to hydrolyse the acetyl phosphate formed (Ruff et al., 2003). ...
The Gram-positive bacteria Rhodococcus opacus ISO-5 and Rhodococcus sp. RHA1 utilized taurine (2-aminoethanesulfonate) as the sole source of carbon or of nitrogen or of sulfur for growth. Different gene clusters and enzymes were active under these different metabolic situations. Under carbon- or nitrogen-limited conditions three enzymes were induced, though to different levels: taurine-pyruvate aminotransferase (Tpa), alanine dehydrogenase (Ald) and sulfoacetaldehyde acetyltransferase (Xsc). The specific activities of these enzymes in R. opacus ISO-5 were sufficient to explain the growth rates under the different conditions. These three enzymes were purified and characterized, and the nature of each reaction was confirmed. Analyses of the genome of Rhodococcus sp. RHA1 revealed a gene cluster, tauR-ald-tpa, putatively encoding regulation and oxidation of taurine, located 20 kbp from the xsc gene and separate from two candidate phosphotransacetylase (pta) genes, as well as many candidate ABC transporters (tauBC). PCR primers allowed the amplification and sequencing of the tauR-ald-tpa gene cluster and the xsc gene in R. opacus ISO-5. The N-terminal sequences of the three tested proteins matched the derived amino acid sequences of the corresponding genes. The sequences of the four genes found in each Rhodococcus strain shared high degrees of identity (>95% identical positions). RT-PCR studies proved transcription of the xsc gene when taurine was the source of carbon or of nitrogen. Under sulfur-limited conditions no xsc mRNA was generated and no Xsc was detected. Taurine dioxygenase (TauD), the enzyme catalysing the anticipated desulfonative reaction when taurine sulfur is assimilated, was presumed to be present because oxygen-dependent taurine disappearance was demonstrated with taurine-grown cells only. A putative tauD gene (with three other candidates) was detected in strain ISO-5. Regulation of the different forms of metabolism of taurine remains to be elucidated.
... Alanine dehydrogenase [EC 1.4.1.4] regenerates pyruvate and releases the ammonium ion (Laue & Cook, 2000a). A sulphoacetaldehyde acetyltransferase [2006 ), 1, pp. 74 -79 (Ruff et al., 2003). ...
... There is very little gene-specific sequence information available to identify B. wadsworthia. The DSR shares more than 80% amino acid identity with other DSRs (Laue et al., 2001), and the alanine dehydrogenase shares more than 60% identity with other alanine dehydrogenases (Laue & Cook, 2000a). In contrast, Tpa usually has low similarity to other aminotransferases with known function (Laue & Cook, 2000b; SupplementaryFig. ...
The bile-resistant, strictly anaerobic bacterium Bilophila wadsworthia is found in human faecal flora, in human infections and in environmental samples. A specific PCR primer set for the gene encoding the first metabolic enzyme in the degradative pathway for taurine in B. wadsworthia, taurine:pyruvate aminotransferase (tpa), was developed and tested. In addition, enrichment cultures were started from faecal samples of primates and felines and shown to contain B. wadsworthia. These were subcultured on agar media and then identified by PCR fingerprinting. PCR for tpa was successful in all positive enrichment cultures and showed no amplification signal in a variety of other bacterial species. Therefore, this PCR method could be a promising tool for rapid detection of B. wadsworthia in biological samples.
... Fig. 1 Overview of the taurine desulfonation pathway in B. wadsworthia 3.1.6, as revealed previously [1,26,27], and of the key enzymes of the pathway enclosed in bacterial microcompartments (BMCs), as inferred from the results of this study (A) Illustration of the taurine degradation pathway via isethionate desulfonation by a glycyl radical enzyme and the subsequent conversion of the acetaldehyde released from isethionate to acetate, and of the reduction of the sulfite released to H 2 S by the dissimilatory sulfite reductase complex. The involvement of BMCs in this pathway, as inferred from the results of this study, is also indicated (see main text). ...
Background
Bilophila wadsworthia , a strictly anaerobic, sulfite-reducing bacterium and common member of the human gut microbiota, has been associated with diseases such as appendicitis and colitis. It is specialized on organosulfonate respiration for energy conservation, i.e., utilization of dietary and host-derived organosulfonates, such as taurine (2-aminoethansulfonate), as sulfite donors for sulfite respiration, producing hydrogen sulfide (H 2 S), an important intestinal metabolite that may have beneficial as well as detrimental effects on the colonic environment. Its taurine desulfonation pathway involves the glycyl radical enzyme (GRE) isethionate sulfite-lyase (IslAB), which cleaves isethionate (2-hydroxyethanesulfonate) into acetaldehyde and sulfite.
Results
We demonstrate that taurine metabolism in B. wadsworthia 3.1.6 involves bacterial microcompartments (BMCs). First, we confirmed taurine-inducible production of BMCs by proteomic, transcriptomic and ultra-thin sectioning and electron-microscopical analyses. Then, we isolated BMCs from taurine-grown cells by density-gradient ultracentrifugation and analyzed their composition by proteomics as well as by enzyme assays, which suggested that the GRE IslAB and acetaldehyde dehydrogenase are located inside of the BMCs. Finally, we are discussing the recycling of cofactors in the IslAB-BMCs and a potential shuttling of electrons across the BMC shell by a potential iron-sulfur (FeS) cluster-containing shell protein identified by sequence analysis.
Conclusions
We characterized a novel subclass of BMCs and broadened the spectrum of reactions known to take place enclosed in BMCs, which is of biotechnological interest. We also provided more details on the energy metabolism of the opportunistic pathobiont B. wadsworthia and on microbial H 2 S production in the human gut.
... Pyruvate, required by Tpa, is regenerated by alanine dehydrogenase (AlaDH). In B. wadsworthia RZATAU, Tpa and AlaDH activities have been detected in lysates of taurine-grown cells, and their primary sequences have been identified 34,35 . However, Xsc activity could not be detected in the lysates 1 , and a BLAST search for Xsc revealed no homologs in both of the sequenced Bilophila species, suggesting a distinct pathway for taurine degradation in Bilophila. ...
Bacterial degradation of organosulfonates plays an important role in sulfur recycling, and has been extensively studied. However, this process in anaerobic bacteria especially gut bacteria is little known despite of its potential significant impact on human health with the production of toxic H2S. Here, we describe the structural and biochemical characterization of an oxygen-sensitive enzyme that catalyzes the radical-mediated C-S bond cleavage of isethionate to form sulfite and acetaldehyde. We demonstrate its involvement in pathways that enables C2 sulfonates to be used as terminal electron acceptors for anaerobic respiration in sulfate- and sulfite-reducing bacteria. Furthermore, it plays a key role in converting bile salt-derived taurine into H2S in the disease-associated gut bacterium Bilophila wadsworthia. The enzymes and transporters in these anaerobic pathways expand our understanding of microbial sulfur metabolism, and help deciphering the complex web of microbial pathways involved in the transformation of sulfur compounds in the gut.
... These gene clusters are conserved in all sequenced Bilophila genomes. The first gene cluster encodes the known enzymes taurine:pyruvate aminotransferase (Tpa) and alanine dehydrogenase (Ald), as previously identified in a different B. wadsworthia isolate (27,28). Tpa converts taurine to sulfoacetaldehyde, and Ald regenerates the amino group acceptor for Tpa (Fig. 1B). ...
Significance
This paper describes a pathway for anaerobic bacterial metabolism of taurine (2-aminoethanesulfonate), an abundant substrate in the human intestinal microbiota, by the intestinal bacterium and opportunistic pathogen, Bilophila wadsworthia . This metabolism converts taurine to the toxic metabolite hydrogen sulfide (H 2 S), an activity associated with inflammatory bowel disease and colorectal cancer. A critical enzyme in this pathway is isethionate sulfite-lyase, a member of the glycyl radical enzyme family. This enzyme catalyzes a novel, radical-based C-S bond-cleavage reaction to convert isethionate (2-hydroxyethanesulfonate) to sulfite and acetaldehyde. This discovery improves our understanding of H 2 S production in the human body and may also offer new approaches for controlling intestinal H 2 S production and B. wadsworthia infections.
... The AlaDH of S. anulatus is dimeric with monomeric units of 61 kDa. However, the hexameric AlaDHs with subunits of approximately 40 kDa were most commonly reported from the microbes and these include AlaDHs from Bacillus cereus, S. phaeochromogenes and B. wadsworthia [32,35,36]. A tetramer with subunits of 43 kDa was found in the soya bean nodule bacteroids [37]. ...
Screening of different unexplored species of Streptomyces led to the identification of alanine dehydrogenase (AlaDH) from Streptomyces anulatus. This AlaDH was purified, characterized and was used as a bioreceptor for developing an ammonium biosensor that can detect ammonium ions in water samples. The AlaDH of S. anulatus was a dimer with each monomeric unit of Mol. Wt. 61 kDa. The optimum pH for AlaDH in oxidative deamination and reductive amination was 10 and 8.5 respectively with wide working pH range of 5-11. The optimum temperature was 40 °C in both the reactions with wide working temperature range of 20-50 °C. The enzyme retained more than 85% of its original activity after incubating at 60 °C for 30 min in the presence of DTT. Due to these properties of AlaDH, it was successfully used as a bioreceptor in the ammonium biosensor and the sensor showed linear response in the range of 0.1-300 mM NH4⁺ with the detection limit of 0.01 mM NH4⁺ and response time of 20 s. The sensor was showing good response at wide pH (5-11) and temperature range (20-50 °C) suggesting its usage at ambient and non-ambient conditions. The sensor was successfully validated with Nessler's reagent method by using real water samples.
... From this, it can be released as the ammonium ion by the action of alanine dehydrogenase (EC 1.4.1.4), which has been purified from B. wadsworthia (Laue and Cook 2000b). Toyama et al. (1973) claimed a third enzyme in P. putida, again a pyruvate-dependent transamination of taurine, but the authors renamed it ω-amino acid:pyruvate aminotransferase (e.g. ...
Organosulfonates are widespread in the environment, both as natural products and as xenobiotics; and they generally share the property of chemical stability. A wide range of phenomena has evolved in microorganisms able to utilize the sulfur or the carbon moiety of these compounds; and recent work has centered on bacteria. This Mini-Review centers on bacterial catabolism of the carbon moiety in the C2-sulfonates and the fate of the sulfonate group. Five of the six compounds examined are subject to catabolism, but information on the molecular nature of transport and regulation is based solely on sequencing data. Two mechanisms of desulfonation have been established. First, there is the specific monooxygenation of ethanesulfonate or ethane-1,2-disulfonate. Second, the oxidative, reductive and fermentative modes of catabolism tend to yield the intermediate sulfoacetaldehyde, which is now known to be desulfonated to acetyl phosphate by a thiamin-diphosphate-dependent acetyltransferase. This enzyme is widespread and at least three subgroups can be recognized, some of them in genomic sequencing projects. These data emphasize the importance of acetyl phosphate in bacterial metabolism. A third mechanism of desulfonation is suggested: the hydrolysis of sulfoacetate.
... SDS-PAGE was done with 12% separative gels (Scha¨gger and von Jagow 1987), and proteins were stained with colloidal Coomassie Brilliant Blue G-250 (Neuhoff et al. 1988); a 10-kDa Protein Ladder (Gibco) was used for calibration. Proteins were subject to blotting and Nterminal sequencing, as described elsewhere (Laue and Cook 2000). ...
Comamonas testosteroni T-2 degraded at least eight aromatic compounds via protocatechuate (PCA), whose extradiol ring cleavage to 2-hydroxy-4-carboxymuconate semialdehyde (HCMS) was catalysed by PCA 4,5-dioxygenase (PmdAB). This inducible, heteromultimeric enzyme was purified. It contained two subunits, α (PmdA) and β (PmdB), and the molecular masses of the denatured proteins were 18 kDa and 31 kDa, respectively. PCA was converted stoichiometrically to HCMS with an apparent K
m of 55 μM and at a maximum velocity of 1.5 μkat. Structure–activity-relationship analysis by testing 16 related compounds as substrate for purified PmdAB revealed an absolute requirement for the vicinal diol and for the carboxylate group of PCA. Besides PCA, only 5′-hydroxy-PCA (gallate) induced oxygen uptake. The N-terminal amino acid sequence of each subunit was identical to the corresponding sequences in C. testosteroni BR6020, which facilitated sequencing of the pmdAB genes in strain T-2. Small differences in the amino acid sequence had significant effects on enzyme stability. Several homologues of pmdAB were found in sequence databases. Residues involved in substrate binding are highly conserved among the homologues. Their sequences grouped within the class III extradiol dioxygenases. Based on our biochemical and genetic analyses, we propose a new branch of the heteromultimeric enzymes within that class.
... Hence, Ald performs similar functions in different contexts in M. maripaludis and B. subtilis. Interestingly, in Bilophila wadsworthia, which also appears to have obtained Ald from the class Firmicutes, the same reaction functions in a different pathway, that of taurine degradation (15). There, Ald functions to regenerate pyruvate after a transaminase has transferred an amino group from taurine to pyruvate to form alanine. ...
Among the archaea, Methanococcus maripaludis has the unusual ability to use l- or d-alanine as a nitrogen source. To understand how this occurs, we tested the roles of three adjacent genes encoding homologs
of alanine dehydrogenase, alanine racemase, and alanine permease. To produce mutations in these genes, we devised a method
for markerless mutagenesis that builds on previously established genetic tools for M. maripaludis. The technique uses a negative selection strategy that takes advantage of the ability of the M. maripaludis hpt gene encoding hypoxanthine phosphoribosyltransferase to confer sensitivity to the base analog 8-azahypoxanthine. In addition,
we developed a negative selection method to stably incorporate constructs into the genome at the site of the upt gene encoding uracil phosphoribosyltransferase. Mutants with in-frame deletion mutations in the genes for alanine dehydrogenase
and alanine permease lost the ability to grow on either isomer of alanine, while a mutant with an in-frame deletion mutation
in the gene for alanine racemase lost only the ability to grow on d-alanine. The wild-type gene for alanine dehydrogenase, incorporated into the upt site, complemented the alanine dehydrogenase mutation. Hence, the permease is required for the transport of either isomer,
the dehydrogenase is specific for the l isomer, and the racemase converts the d isomer to the l isomer. Phylogenetic analysis indicated that all three genes had been acquired by lateral gene transfer from the low-moles-percent
G+C gram-positive bacteria.
... by replacing pyruvate with 2-oxoglutarate. Alanine dehydrogenase was assayed by a standard method (Laue & Cook, 2000). Taurine dehydrogenase was assayed with either dichlorophenol indophenol (DCPIP) or beef-heart cytochrome c as the electron acceptor (Brüggemann et al., 2004). ...
Eighteen enrichment cultures with taurine (2-aminoethanesulfonate) as the sole source of combined nitrogen under aerobic conditions were all successful, and 24 pure cultures were obtained. Only three of the cultures yielded an inorganic product, sulfate, from the sulfonate moiety of taurine, and the others were presumed to yield organosulfonates. Sulfoacetate, known from Rhodopseudomonas palustris CGA009 under these conditions, was not detected in any culture, but sulfoacetaldehyde (as a hydrazone derivative) was tentatively detected in the outgrown medium of nine isolates. The compound was stable under these conditions and the identification was confirmed by MALDI-TOF-MS. Most sulfoacetaldehyde-releasing isolates were determined to be strains of Acinetobacter calcoaceticus, and a representative organism, strain SW1, was chosen for further work. A quantitative enzymic determination of sulfoacetaldehyde and its bisulfite addition complex was developed: it involved the NAD-coupled sulfoacetaldehyde dehydrogenase from R. palustris. A. calcoaceticus SW1 utilized taurine quantitatively and concomitantly with growth in, for example, an adipate-salts medium, and the release of sulfoacetaldehyde was stoichiometric. The deamination reaction involved a taurine dehydrogenase. Enrichment cultures to explore the possible release of organophosphonates from the analogous substrate, 2-aminoethanephosphonate, led to 33 isolates, all of which released inorganic phosphate quantitatively.
... The need for an alanine dehydrogenase (Ald) [EC 1.4.1.4] in the hypothesis in Fig. 1 is known (Laue and Cook 2000b;Denger et al. 2004), as are roles for a sulfite dehydrogenase (sulfite oxidoreductase, Sor) and exporters of ammonium and sulfate ions, but candidate genes to encode the latter functions are either absent or unknown (Sor) or have not been confirmed . Thus, despite the logic of the scheme in Fig. 1, and the utilization of taurine, there is no experimental support for a sometimes tenuous hypothesis (e.g., 23% identity with a confirmed ortholog). ...
A largely untested hypothesis for the bacterial dissimilation of taurine was explored in Silicibacter pomeroyi DSS-3, whose genome has been sequenced. Substrate-specific transcription of candidate genes encoding taurine uptake and dissimilation (tauABC, tpa, ald, xsc, pta) was found, which corresponded to the induction of Tpa, Ald, Xsc and Pta, that was observed.
... Further hypotheses on the dissimilation of N-methyltaurine involved the nature of removal of the methyl group, possibly by its oxygenation to yield taurine, followed by either taurine : pyruvate aminotransferase (EC 2.6.1.77) with alanine dehydrogenase (Laue & Cook, 2000;Denger et al., 2004a) to yield sulfoacetaldehyde, or by taurine : ferricytochrome-c oxidoreductase (deaminating) (taurine dehydrogenase, presumably EC 1.4.2.-) to yield sulfoacetaldehyde (Brüggemann et al., 2004). Alternatively, the reaction might proceed by hydrolytic removal of the methylamino moiety to yield sulfoacetaldehyde, analogous to reactions (e.g. ...
Selective enrichments yielded bacterial cultures able to utilize the osmolyte N-methyltaurine as sole source of carbon and energy or as sole source of fixed nitrogen for aerobic growth. Strain MT1, which degraded N-methyltaurine as a sole source of carbon concomitantly with growth, was identified as a strain of Alcaligenes faecalis. Stoichiometric amounts of methylamine, whose identity was confirmed by matrix-assisted, laser-desorption ionization time-of-flight mass spectrometry, and of sulfate were released during growth. Inducible N-methyltaurine dehydrogenase, sulfoacetaldehyde acetyltransferase (Xsc) and a sulfite dehydrogenase could be detected. Taurine dehydrogenase was also present and it was hypothesized that taurine dehydrogenase has a substrate range that includes N-methyltaurine. Partial sequences of a tauY-like gene (encoding the putative large component of taurine dehydrogenase) and an xsc gene were obtained by PCR with degenerate primers. Strain N-MT utilized N-methyltaurine as a sole source of fixed nitrogen for growth and could also utilize the compound as sole source of carbon. This bacterium was identified as a strain of Paracoccus versutus. This organism also expressed inducible (N-methyl)taurine dehydrogenase, Xsc and a sulfite dehydrogenase. The presence of a gene cluster with high identity to a larger cluster from Paracoccus pantotrophus NKNCYSA, which is now known to dissimilate N-methyltaurine via Xsc, allowed most of the overall pathway, including transport and excretion, to be defined. N-Methyltaurine is thus another compound whose catabolism is channelled directly through sulfoacetaldehyde.
... Taurine : pyruvate aminotransferase (Tpa) was assayed discontinuously as the pyruvate-dependent disappearance of taurine concomitant with the formation of alanine (Brüggemann et al., 2004 ). Alanine dehydrogenase (Ald) was routinely measured photometrically as the reduction of NAD + (Laue & Cook, 2000a); S. pomeroyi DSS-3 (Denger et al., 2006) served as a positive control. Sulfoacetaldehyde acetyltransferase (Xsc) was assayed by GC as the ThDP-and phosphate-dependent release of acetate after acidification to hydrolyse the acetyl phosphate formed, or assayed as the formation of sulfite (Ruff et al., 2003). ...
A degradative pathway for taurine (2-aminoethanesulfonate) in Rhodobacter sphaeroides 2.4.1 was proposed by Brüggemann et al. (2004) (Microbiology 150, 805-816) on the basis of a partial genome sequence. In the present study, R. sphaeroides 2.4.1 was found to grow exponentially with taurine as the sole source of carbon and energy for growth. When taurine was the sole source of nitrogen in succinate-salts medium, the taurine was rapidly degraded, and most of the organic nitrogen was excreted as the ammonium ion, which was then utilized for growth. Most of the enzymes involved in dissimilation, taurine dehydrogenase (TDH), sulfoacetaldehyde acetyltransferase (Xsc) and phosphate acetyltransferase (Pta), were found to be inducible, and evidence for transcription of the corresponding genes (tauXY, xsc and pta), as well as of tauKLM, encoding the postulated TRAP transporter for taurine, and of tauZ, encoding the sulfate exporter, was obtained by reverse-transcription PCR. An additional branch of the pathway, observed by Novak et al. (2004) (Microbiology 150, 1881-1891) in R. sphaeroides TAU3, involves taurine : pyruvate aminotransferase (Tpa) and a presumptive ABC transporter (NsbABC). No evidence for a significant role of this pathway, or of the corresponding alanine dehydrogenase (Ald), was obtained for R. sphaeroides 2.4.1. The anaplerotic pathway needed under these conditions in R. sphaeroides 2.4.1 seems to involve malyl-CoA lyase, which was synthesized inducibly, and not malate synthase (GlcB), whose presumed gene was not transcribed under these conditions.
... The assays of SafD and Tpa involved the same buffer, and they could be combined to allow conversion of taurine to sulfoacetate. Alanine dehydrogenase (Ald) was assayed spectrophotometrically as reduction of NAD + (Laue and Cook 2000b): the positive control was from Rhodococcus opacus ISO-5 (Denger et al. 2004a). Sulfoacetaldehyde acetyltransferase (Xsc) [EC 2.3.3.15] was assayed as release of sulfite from sulfoacetaldehyde with the enzyme from Cupriavidus necator H16 as positive control (Ruff et al. 2003;Weinitschke et al. 2007). ...
Taurine (2-aminoethanesulfonate) is a widespread natural product whose nitrogen moiety was recently shown to be assimilated by bacteria, usually with excretion of an organosulfonate via undefined novel pathways; other data involve transcriptional regulator TauR in taurine metabolism. A screen of genome sequences for TauR with the BLAST algorithm allowed the hypothesis that the marine gammaproteobacterium Neptuniibacter caesariensis MED92 would inducibly assimilate taurine-nitrogen and excrete sulfoacetate. The pathway involved an ABC transporter (TauABC), taurine:pyruvate aminotransferase (Tpa), a novel sulfoacetaldehyde dehydrogenase (SafD) and exporter(s) of sulfoacetate (SafE) (DUF81). Ten candidate genes in two clusters involved three sets of paralogues (for TauR, Tpa and SafE). Inducible Tpa and SafD were detected in cell extracts. SafD was purified 600-fold to homogeneity in two steps. The monomer had a molecular mass of 50 kDa (SDS-PAGE); data from gel filtration chromatography indicated a tetrameric native protein. SafD was specific for sulfoacetaldehyde with a K (m)-value of 0.12 mM. The N-terminal amino acid sequence of SafD confirmed the identity of the safD gene. The eight pathway genes were transcribed inducibly, which indicated expression of the whole hypothetical pathway. We presume that this pathway is one source of sulfoacetate in nature, where this compound is dissimilated by many bacteria.
Unnatural amino acids are unique building blocks in modern medicinal chemistry as they contain an amino and a carboxylic acid functional group, and a variable side chain. Synthesis of pure unnatural amino acids can be made through chemical modification of natural amino acids or by employing enzymes that can lead to novel molecules used in the manufacture of various pharmaceuticals. The NAD+ -dependent alanine dehydrogenase (AlaDH) enzyme catalyzes the conversion of pyruvate to L-alanine by transferring ammonium in a reversible reductive amination activity. Although AlaDH enzymes have been widely studied in terms of oxidative deamination activity, reductive amination activity studies have been limited to the use of pyruvate as a substrate. The reductive amination potential of heterologously expressed, highly pure Thermomicrobium roseum alanine dehydrogenase (TrAlaDH) was examined with regard to pyruvate, α-ketobutyrate, α-ketovalerate and α-ketocaproate. The biochemical properties were studied, which included the effects of 11 metal ions on enzymatic activity for both reactions. The enzyme accepted both derivatives of L-alanine (in oxidative deamination) and pyruvate (in reductive amination) as substrates. While the kinetic KM values associated with the pyruvate derivatives were similar to pyruvate values, the kinetic kcat values were significantly affected by the side chain increase. In contrast, KM values associated with the derivatives of L-alanine (L-α-aminobutyrate, L-norvaline, and L-norleucine) were approximately two orders of magnitude greater, which would indicate that they bind very poorly in a reactive way to the active site. The modeled enzyme structure revealed differences in the molecular orientation between L-alanine/pyruvate and L-norleucine/α-ketocaproate. The reductive activity observed would indicate that TrAlaDH has potential for the synthesis of pharmaceutically relevant amino acids.
A novel alanine dehydrogenase (ADH; EC.1.4.1.1) with high pyruvate reduced activity was isolated from Helicobacter aurati and expressed in Escherichia coli BL21 (DE3). The optimum pH of the reduction and oxidation reaction were 8.0 and 9.0, respectively, and the optimum temperature was 55 °C. With pyruvate and alanine as substrates, the specific activity of HAADH1 were 268 U·mg⁻¹ and 26 U·mg⁻¹, respectively. HAADH1 had a prominent substrate specificity for alanine (Km = 2.23 mM, kcat/Km = 8.1 s⁻¹·mM⁻¹). In the reduction reaction, HAADH1 showed the highest substrate affinity for pyruvate (Km = 0.56 mM, kcat/Km = 364 s⁻¹·mM⁻¹). Compared to pyruvate, oxaloacetic acid, 2-ketobutyric acid, 3-fluoropyruvate, α-ketoglutaric acids, glyoxylic acid showed a residual activity of 93.30%, 8.93%, 5.62%, 2.57%, 2.51%, respectively. Phylogenetic tree analysis showed that this is a new type of ADH which have a low sequence similarity to available ADH reported in references. 3-Fluoropyruvate was effectively reduced to 3-fluoro-L-alanine by whole-cell catalysis.
Bacteria utilize diverse biochemical pathways for the degradation of the pyrimidine ring. The function of the pathways studied to date has been the release of nitrogen for assimilation. The most widespread of these pathways is the reductive pyrimidine catabolic pathway, which converts uracil into ammonia, carbon dioxide and β-alanine. Here we report the characterization of a β-alanine:pyruvate aminotransferase (PydD2), and a NAD ⁺ -dependent malonic semialdehyde dehydrogenase (MSDH), from a reductive pyrimidine catabolism gene cluster in Bacillus megaterium . Together, these enzymes convert β-alanine into acetyl-CoA, a key intermediate in carbon and energy metabolism. We demonstrate the growth of B. megaterium in defined medium with uracil as its sole carbon and energy source. Homologs of PydD2 and MSDH are found in association with reductive pyrimidine pathway genes in many Gram positive bacteria in the order Bacillales. Our study provides a basis for further investigations of the utilization of pyrimidines as a carbon and energy source by bacteria.
Importance Pyrimidine has wide occurrence in natural environments, where bacteria use it as nitrogen and carbon source for growth. Detailed biochemical pathways have been investigated with focus mainly on nitrogen assimilation in the past decades. Here we report the discovery and characterization of two important enzymes, PydD2 and MSDH, which constitute an extension for the reductive pyrimidine catabolic pathway. These two enzymes, prevalent in Bacillales based on our bioinformatics studies, allow stepwise conversion of β-alanine, a previous “end product” of the reductive pyrimidine degradation pathway to acetyl-CoA, as carbon and energy source.
Alanine dehydrogenase (AlaDH) (E.C.1.4.1.1) is a microbial enzyme that catalyzes a reversible conversion of L-alanine to pyruvate. Inter-conversion of alanine and pyruvate by AlaDH is central to metabolism in microorganisms. Its oxidative deamination reaction produces pyruvate which plays a pivotal role in the generation of energy through the tricarboxylic acid cycle for sporulation in the microorganisms. Its reductive amination reaction provides a route for the incorporation of ammonia and produces L-alanine which is required for synthesis of the peptidoglycan layer, proteins, and other amino acids. Also, AlaDH helps in redox balancing as its deamination/amination reaction is linked to the reduction/oxidation of NAD⁺/NADH in microorganisms. AlaDH from a few microorganisms can also reduce glyoxylate into glycine (aminoacetate) in a nonreversible reaction. Both its oxidative and reductive reactions exhibit remarkable applications in the pharmaceutical, environmental, and food industries. The literature addressing the characteristics and applications of AlaDH from a wide range of microorganisms is summarized in the current review.
A study of the properties of L-alanine dehydrogenase of Trichoderma viride was undertaken. Maximal enzyme activity occurred at pH 8 for reductive amination of pyruvate, and at pH 9.5 for oxidative deamination of L-alanine at a temperature of 50°C. Maximum velocity of the reductive amination reaction was ten times greater than that of the oxidative deamination reaction. The Km values for pyruvate, NH4+, NADH, L-alanine and NAD+ were 17.2, 166, 0.7, 6.06 and 7.14mM respectively. The enzyme was not inhibited by EDTA, suggesting that no metal cation is participation in enzyme catalysis. This is supported by the finding that none of the tested metal salts activated the enzyme. Alternatively, the enzyme activity was inhibited by Fe2+, Mg2+, Ca2+, Cu2+, and Mn2+. SH groups don't seem to play a role in the catalytic action of the enzyme, as addition of iodoacetate or reduced glutathione did not effect the enzyme activity. Stability of the enzyme under different conditions was investigated.
The recombinant alanine dehydrogenase (ADH) from E. coli containing Thermus caldophilus ADH was purified to homogeneity from a cell-free extract. The enzyme was purified 38-fold with a yield of 68% from the starting cell-free extract. The purified enzyme gave a single band in polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 45 kDa. The pH optimum was 8.0 for reductive amination of pyruvate and 12.0 for oxidative deamination of L-alanine. The enzyme was stable up to 70°C. The activity of the enzyme was inhibited by 1 mM Zn2+, 20% hexane, and 20% CHCl3. However, 10 mM Mg2+ and 40% propanol had no effect on the enzyme activity. The Michaelis constants (Km) for the substrates were 50 μM for NADH, 0.2 mM for pyruvate, 39.4 mM for NH4+, 2.6 mM for L-alanine, and 1.8 mM for NAD+.
Alanine dehydrogenase has not yet been purified from any fungal sources. The aims of this study was to isolate alanine dehydrogenase from Thielaviopsis paradoxa. The biochemical kinetic properties of the enzyme like Km, Vmax, optimum Temperature and pH were also determined. In this study, effect of certain inhibitors on the enzyme was tested. Alanine dehydrogenase (EC 1.4.1.1), an enzyme that catalyzes the reversible deamination of L-alanine in the presence of NAD +, was extracted and characterized from Thielaviopsis paradoxa, a pathogenic fungi for the date palm (Phoenix dactylifera). The enzyme showed maximal activity at pH 9.5 for the deamination of L-alanine and at pH 8.5 for the amination of pyruvate. It was active in the presence of both NAD + and NADP + as coenzymes for the deamination of L-alanine. In addition, the enzyme was not absolutely specific to L-alanine as a substrate in the deamination of L-alanine. L-alanine dehydrogenase had Km value for L-alanine (1.35 mM), for NAD + 0.274 mM, NADH (0.197 mM), pyruvate (8.16 mM) and ammonia (33.5 mM). It was inhibited by Zn 2+, CO 2+ and by iodoacetate. Activation by using reduced glutathione and DTT are suggestive of sulfhydryl group participation in enzyme activity.
Hypotaurine (HT, 2-aminoethane-sulfinate) is known to be utilized by bacteria as a sole source of carbon, nitrogen and energy for growth, as is taurine (2-aminoethane-sulfonate), however, the corresponding HT-degradation pathway remained undefined. Genome-sequenced Paracoccus denitrificans PD1222 utilized HT (and taurine) quantitatively for heterotrophic growth and released the HT-sulfur as sulfite (and sulfate), and HT-nitrogen as ammonium. Enzyme assays with cell-free extracts suggested that a HT-inducible HT:pyruvate aminotransferase (Hpa) catalyzes the deamination of HT in an initial reaction step. Partial purification of the Hpa activity and peptide fingerprinting-mass spectrometry (PF-MS) identified the Hpa-candidate gene; it encoded an archetypal taurine:pyruvate aminotransferase (Tpa). The same gene-product was identified via differential PAGE and PF-MS, as well as the gene of a strongly HT-inducible aldehyde dehydrogenase (Adh). Both genes were overexpressed in E. coli. The overexpressed, purified Hpa/Tpa showed HT:pyruvate-aminotransferase activity. Alanine, acetaldehyde, and sulfite, were identified as the reaction products, but not sulfinoacetaldehyde; the reaction of Hpa/Tpa with taurine yielded sulfoacetaldehyde, which is stable. The overexpressed, purified Adh oxidized the acetaldehyde generated during the Hpa reaction to acetate in an NAD(+)-dependent reaction. Based on these results, the following degradation pathway for HT in strain PD1222 can be depicted. The identified aminotransferase converts HT to sulfinoacetaldehyde, which desulfinates spontaneously to acetaldehyde and sulfite; the inducible aldehyde dehydrogenase oxidizes acetaldehyde to yield acetate, which is metabolized, and sulfite, that is excreted.
Taurine (2-aminoethanesulfonate), isethionate (2-hydroxyethanesulfonate) and sulfoacetate are natural C2 sulfonates which exist in the environment and are to our current knowledge solely degraded by microorganisms which are able to cleave the chemically stable C-sulfonate bond. Anaerobic and aerobic degradation of taurine is well investigated in diverse (marine and terrestrial) bacteria. The aerobic dissmilation of taurine proceeds via the central intermediate sulfoacetaldehyde (SAA) and subsequent desulfonation by sulfoacetaldehyde acetyltransferase (Xsc) to acetylphosphate and sulfite. Acetylphosphate is then metabolized (by two different possible pathways, e.g. by Pta, phosphate acetyltransferase) to acetyl-CoA and thereby channeled into central metabolism. Sulfite is, for the purpose of detoxification, oxidized to sulfate by sulfite dehydrogenase. It is a generally accepted hypothesis that the degradative pathways of isethionate and sulfoacetate converge at SAA with other (C2) sulfonates. In this study, this hypothesis was confirmed in the bacterium Cupriavidus necator H16: the organism was able to utilize taurine, isethionate and sulfoacetate as a sole source of carbon and energy for growth, and it excreted stoichiometric amounts of sulfate into the growth medium. Inducible enzyme activities were measured during growth with each of the three sulfonates. Additionally, transcription experiments (RT-PCR, reverse transcription PCR) with the appropriate genes showed inducible transcription of each of the genes in mRNA extracted from sulfoacetate-grown cells. Furthermore, a transport protein (TauE) was presumed to represent a sulfite exporter wich is responsible for the translocation of sulfite into the periplasm, its site of oxidation by SorAB. In addition, the previously uncharacterized initial steps of isethionate and sulfoacetate, leading to SAA, were investigated: The microbial dissimilation of isethionate is presumed to occur via inducible isethionate dehydrogenase (IseJ) in marine and terrestrial bacteria. A gene cluster was found which presumably encodes the isethionate degradative genes. The gene products include a putative transcriptional regulator (IseR), isethionate dehydrogenase (IseJ) as well as a transport system (IseU in terrestrial bacteria, IseKLM in marine bacteria). The microbial degradation of sulfoacetate in C. necator H16 proceeds via an ATP-dependent activation of sulfoacetate to the novel CoA-ester sulfoacetyl-CoA. This intermediate was identified by MALDI-TOF mass spectrometry. The enzyme catalyzing this reaction was sulfoacetate-CoA ligase (SauT) which could be measured in a discontinuous enzyme assay at the HPLC. In the next step, sulfoacetyl-CoA was converted to SAA by sulfoacetaldehyde dehydrogenase (SauS). Both SauT and SauS were inducibly active during growth with sulfoacetate. SauS was purified to homogeneity, characterized and assigned to the coding gene (H16_A2747) by PMF (peptide mass fingerprinting). The gene sauS was part of a cluster consisting of four genes encoding regulation (SauR), activation (SauT), reduction (SauS) and transport (SauU). The catabolic genes sauSTU were inducibly transcribed during growth with sulfoacetate, as confirmed by RT-PCR. In addition, the involvement of the genes in sulfoacetate degradation was proven by site-directed mutagenesis. By comparative genomics, 25 microorganisms, both marine and terrestrial, were found to contain sulfoacetate gene clusters. Thereby, a different putative activation enzyme (heteromeric sulfoacetate-CoA ligase SauPQ), different types of regulators (SauI, SauV) and an alternative transport system (TTT, tripartite tricarboxylate transporter, SauFGH) for sulfoacatate were discovered. Some of these variants of the newly discovered sulfoacetate degradation pathway were investigated in e.g. Roseovarius nubinhibens ISM and Oligotropha carboxidovorans OM5 by means of growth experiments, enzyme activity tests and RT-PCR. Taurin, Isethionat und Sulfoacetat sind natürlich vorkommende C2-Sulfonate, die in der Umwelt vorliegen und dort nach heutigem Wissensstand ausschließlich von Mikroorganismen abgebaut werden können, da nur diese in der Lage sind, die chemisch stabile Kohlenstoff-Sulfonat-Bindung zu spalten. Der aerobe sowie anaerobe Abbau von Taurin in verschiedenen Bakterien wurde bereits weitgehend geklärt. Die aerobe Dissimilation von Taurin verläuft immer über das zentrale Intermediat Sulfoacetaldehyd (SAA) und dessen anschließende Desulfonierung zu Acetylphosphat und Sulfit, katalysiert durch die Sulfoacetaldehyd-Acetyltransferase (Xsc). Das dabei entstehende Acetylphosphat wird (auf verschiedenen möglichen Reaktionswegen) zu Acetyl-CoA umgesetzt (beispielsweise durch Pta, Phosphat-Acetyltransferase) und kann somit in den zentralen Stoffwechsel eingeschleust werden. Das ebenfalls entstandene Sulfit wird durch eine Sulfit-Dehydrogenase zu Sulfat oxidiert. Von Isethionat sowie Sulfoacetat wird seit einigen Jahren angenommen, dass sie wie Taurin ebenfalls über SAA abgebaut werden. In der vorliegenden Arbeit wurde im Bakterium Cupriavidus necator H16 diese Vermutung bestätigt: Das Bakterium war in der Lage, Taurin, Isethionat und Sulfoacetat als jeweils alleinige Kohlenstoffquelle zu verwenden und schied in stöchiometrischem Verhältnis Sulfat aus. Induzierbare Enzymaktivitäten sowie Transkriptions-Experimente mit den jeweiligen Genen zeigten, dass alle drei Sulfonate über SAA abgebaut wurden. Es wurde weiterhin ein potentielles Transportproteinen (TauE) für Sulfit gefunden, welches vermutlich Sulfit aus dem Cytoplasma in das Periplasma transportiert, wo es durch die Sulfit-Dehydrogenase zu Sulfat oxidiert wird. Als weitere Themen der vorliegenden Doktorarbeit wurden die initialen, bislang unbekannten Schritte des Abbaus von Sulfoacetat sowie Isethionat auf dem Weg zum gemeinsamen Zwischenprodukt (SAA) untersucht. Der Isethionat-Abbau verläuft in terrestrischen sowie marinen Bakterien vermutlich über eine induzierbare Isethionat-Dehydrogenase (IseJ). Mit Hilfe von RT-PCR wurden Gene identifiziert, die vermutlich die für den Isethionat-Abbau benötigten Enzyme kodieren und in allen Isethionat-verwertenden Bakterienstämmen ein definiertes Cluster bilden. Diese Gene kodieren vermutlich einen Regulator (IseR), die Isethionat-Dehydrogenase (IseJ) sowie verschiedene Transport-systeme (IseU in terrestrischen Bakterien, IseKLM in marinen Bakterien). Der Sulfoacetat-Abbau verläuft in Cupriavidus necator H16 über eine ATP-abhängige Aktivierung von Sulfoacetat zu Sulfoacetyl-CoA, einem neuartigen CoA-Ester. Dieser CoA-Ester wurde mittels HPLC gemessen und mit Hilfe von MALDI-TOF-Massenspektrometrie als Sulfoacetyl-CoA identifiziert. Das katalysierende Enzym, Sulfoacetat-CoA-Ligase (SauT), wurde in einem diskontinuierlichen Enzymtest mittels HPLC nachgewiesen. Im nächsten Schritt wurde Sulfoacetyl-CoA von der Sulfoacetaldehyd-Dehydrogenase (SauS) zu Sulfoacetaldehyd umgesetzt. Beide Enzyme waren nur bei Wachstum mit Sulfoacetat aktiv. SauS wurde gereinigt und charakterisiert, und mittels PMF (Peptide Mass Fingerprint) konnte das Enzym seinem Genlokus (H16_A2747) zugeordnet werden. Das Gen sauS ist Teil eines Genclusters bestehend aus 4 Genen. Deren Genprodukte umfassen einen transkriptionellen Regulator (SauR), die Sulfoacetaldehyd-Dehydrogenase (SauS), ein als Acetat-CoA-Ligase annotiertes Protein, welches wir für die Sulfoacetat-CoA-Ligase (SauT) halten, sowie ein MFS-Transportprotein (SauU), das den Sulfoacetat-Transporter darstellt. Die katabolischen Gene sauSTU waren induzierbar transkribiert, wie mittels RT-PCR bestätigt. Die Beteiligung dieser drei Gene wurde weiterhin durch gerichtete Deletionsmutagenese bewiesen. Durch Vergleich von Sulfoacetat-Genclustern in 25 verschiedenen Genom-sequenzierten marinen sowie terrestrischen Bakterien wurden alternative Transportsysteme (TTT, tripartite tricarboxylate transporter, SauFGH), Regulatoren (SauI, SauV) sowie eine heteromere Variante der Sulfoacetat-CoA-Ligase (SauPQ) gefunden. Einige dieser Variationen (SauFGH, SauPQ) wurden in Roseovarius nubinhibens ISM sowie Oligotropha carboxidovorans OM5 mittels Wachstumsversuchen, Enzymaktivitätstests sowie RT-PCR untersucht und bestätigt.
A dissimilatory sulfite reductase (DSR) was purified from the anaerobic, taurine-degrading bacterium Bilophila wadsworthia RZATAU to apparent homogeneity. The enzyme is involved in energy conservation by reducing sulfite, which is formed during
the degradation of taurine as an electron acceptor, to sulfide. According to its UV-visible absorption spectrum with maxima
at 392, 410, 583, and 630 nm, the enzyme belongs to the desulfoviridin type of DSRs. The sulfite reductase was isolated as
an α2β2γn(n ≥ 2) multimer with a native size of 285 kDa as determined by gel filtration. We have sequenced the genes encoding the α and
β subunits (dsrA and dsrB, respectively), which probably constitute one operon. dsrAand dsrB encode polypeptides of 49 (α) and 54 kDa (β) which show significant similarities to the homologous subunits of other DSRs.
The dsrB gene product of B. wadsworthiais apparently a fusion protein of dsrB anddsrD. This indicates a possible functional role of DsrD in DSR function because of its presence as a fusion protein as an integral
part of the DSR holoenzyme in B. wadsworthia. A phylogenetic analysis using the available Dsr sequences revealed thatB. wadsworthia grouped with its closest 16S rDNA relative Desulfovibrio desulfuricans Essex 6.
A novel alanine dehydrogenase (AlaDH) showing no significant amino acid sequence homology with previously known bacterial
AlaDHs was purified to homogeneity from the soluble fraction of the hyperthermophilic archaeon Archaeoglobus fulgidus. AlaDH catalyzed the reversible, NAD+-dependent deamination of l-alanine to pyruvate and NH4+. NADP(H) did not serve as a coenzyme. The enzyme is a homodimer of 35 kDa per subunit. The Km values for l-alanine, NAD+, pyruvate, NADH, and NH4+ were estimated at 0.71, 0.60, 0.16, 0.02, and 17.3 mM, respectively. The A. fulgidus enzyme exhibited its highest activity at about 82°C (203 U/mg for reductive amination of pyruvate) yet still retained 30%
of its maximum activity at 25°C. The thermostability of A. fulgidus AlaDH was increased by more than 10-fold by 1.5 M KCl to a half-life of 55 h at 90°C. At 25°C in the presence of this salt
solution, the enzyme was ∼100% stable for more than 3 months. Closely related A. fulgidus AlaDH homologues were found in other archaea. On the basis of its amino acid sequence, A. fulgidus AlaDH is a member of the ornithine cyclodeaminase-μ-crystallin family of enzymes. Similar to the μ-crystallins, A. fulgidus AlaDH did not exhibit any ornithine cyclodeaminase activity. The recombinant human μ-crystallin was assayed for AlaDH activity,
but no activity was detected. The novel A. fulgidus gene encoding AlaDH, AF1665, is designated ala.
Quantitative utilization of L-cysteate (2-amino-3-sulphopropionate) as the sole source of carbon and energy for growth of the aerobic, marine bacterium Silicibacter pomeroyi DSS-3(T) was observed. The sulphonate moiety was recovered in the medium largely as sulphite, and the appropriate amount of the ammonium ion was also observed. Genes [suyAB (3-sulpholactate sulpho-lyase)] encoding the known desulphonation reaction in cysteate degradation were absent from the genome, but a homologue of a putative sulphate exporter gene (suyZ) was found, and its neighbour, annotated as a D-cysteine desulphhydrase, was postulated to encode pyridoxal 5'-phosphate-coupled L-cysteate sulpho-lyase (CuyA), a novel enzyme. Inducible CuyA was detected in cysteate-grown cells. The enzyme released equimolar pyruvate, sulphite and the ammonium ion from L-cysteate and was purified to homogeneity by anion-exchange, hydrophobic-interaction and gel-filtration chromatography. The N-terminal amino acid sequence of this 39-kDa subunit confirmed the identification of the cuyA gene. The native enzyme was soluble and homomultimeric. The K(m)-value for L-cysteate was high (11.7 mM) and the enzyme also catalysed the D-cysteine desulphhydrase reaction. The gene cuyZ, encoding the putative sulphite exporter, was co-transcribed with cuyA. Sulphite was exported despite the presence of a ferricyanide-coupled sulphite dehydrogenase. CuyA was found in many bacteria that utilize cysteate.
The gram-negative anaerobic gut bacterium Bilophila wadsworthia is the third most common isolate in perforated and gangrenous appendicitis, being also found in a variety of other infections. This organism performs a unique kind of anaerobic respiration in which taurine, a major organic solute in mammals, is used as a source of sulphite that serves as terminal acceptor for the electron transport chain. We show here that molecular hydrogen, one of the major products of fermentative bacteria in the colon, is an excellent growth substrate for B. wadsworthia. We have quantified the enzymatic activities associated with the oxidation of H(2), formate and pyruvate for cells obtained in different growth conditions. The cell extracts present high levels of hydrogenase activity, and up to five different hydrogenases can be expressed by this organism. One of the hydrogenases appears to be constitutive, whereas the others show differential expression in different growth conditions. Two of the hydrogenases are soluble and are recognised by antibodies against a [FeFe] hydrogenase of a sulphate reducing bacterium. One of these hydrogenases is specifically induced during fermentative growth on pyruvate. Another two hydrogenases are membrane-bound and show increased expression in cells grown with hydrogen. Further work should be carried out to reveal whether oxidation of hydrogen contributes to the virulence of B. wadsworthia.
L-Alanine dehydrogenase from Bacillus subtilis was inactivated with two different lysine-directed chemical reagents, i.e. 2,4,6-trinitrobenzenesulfonic acid and N-succinimidyl 3-(2-pyridyldithio)propionate. In both cases, the inactivation followed pseudo first-order kinetics, with a
1:1 stoichiometric ratio between the reagent and the enzyme subunits. Partial protection of the active site from inactivation
could be obtained by each of the substrates, NADH or pyruvate, but complete protection could only be achieved in the presence
of the ternary complex E·;NADH·;pyruvate. The nucleotide analogue of NADH, 5′-(p-(fluorosulfonyl)benzoyl)adenosine was also used for affinity labeling of the enzyme active site.
Differential peptide mapping, performed both in the presence and in the absence of the substrates, followed by reversed phase
high performance liquid chromatography separation, diode-array analysis, mass spectrometry, and N-terminal sequencing of the
resulting peptides, allowed the identification of lysine 74 in the active site of the enzyme. This residue, which is conserved
among all L-alanine dehydrogenases, is most likely the residue previously postulated to be necessary for the binding of pyruvate in the
active site.
Surprisingly, this residue and the surrounding conserved residues are not found in amino acid dehydrogenases like glutamate,
leucine, phenylalanine, or valine dehydrogenases, suggesting that A-stereospecific amino acid dehydrogenases such as L-alanine dehydrogenase could have evolved apart from the B-stereospecific amino acid dehydrogenases.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic, and statistical refinements permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is described for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position Specific Iterated BLAST (PSLBLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities.
A gene encoding a protein antigen from Mycobacterium tuberculosis with a molecular weight of 40,000 has been sequenced. On the basis of sequence homology and functional analyses, we demonstrated that the protein is an L-alanine dehydrogenase (EC 1.4.1.1). The enzyme was demonstrated in M. tuberculosis and Mycobacterium marinum but not in Mycobacterium bovis BCG. The enzyme may play a role in cell wall synthesis because L-alanine is an important constituent of the peptidoglycan layer. Although no consensus signal sequence was identified, we found evidence which suggests that the enzyme is secreted across the cell membrane. The enzyme was characterized and purified by chromatography, thus enabling further studies of its role in virulence and interaction with the immune system of M. tuberculosis-infected individuals.
Strongly catalase-positive Gram-negative anaerobic rods were isolated from approximately half of all intra-abdominal specimens received from patients with gangrenous and perforated appendicitis, and subsequently also from normal faecal specimens. The organism was originally detected on Bacteroides-bile-aesculin (BBE) agar, and grew slowly on non-selective anaerobic media containing blood. It was stimulated by bile and differed from other known genera by being urease- and catalase-positive, and by reducing nitrate. It did not reduce sulphate. Other anaerobic Gram-negative rods showed no homology by DNA dot-blot hybridization. The thermal melting profile of chromosomal DNA showed 39-40 mol% G + C. The whole-cell fatty acid methyl ester profile included cyclic and branched long-chain acids, and differed from those of all other anaerobes that have been tested. beta-Lactamase was not detected. The name Bilophila wadsworthia gen. nov., sp. nov. is proposed for this organism.
In the phototrophic nonsulfur bacterium Rhodobacter capsulatus E1F1, L-alanine dehydrogenase aminating activity functions as an alternative route for ammonia assimilation when glutamine synthetase is inactivated. L-Alanine dehydrogenase deaminating activity participates in the supply of organic carbon to cells growing on L-alanine as the sole carbon source. L-Alanine dehydrogenase is induced in cells growing on pyruvate plus nitrate, pyruvate plus ammonia, or L-alanine under both light-anaerobic and dark-heterotrophic conditions. The enzyme has been purified to electrophoretic and immunological homogeneity by using affinity chromatography with Red-120 agarose. The native enzyme was an oligomeric protein of 246 kilodaltons (kDa) which consisted of six identical subunits of 42 kDa each, had a Stokes' radius of 5.8 nm, an s20.w of 10.1 S, a D20,w of 4.25 x 10(-11) m2 s-1, and a frictional quotient of 1.35. The aminating activity was absolutely specific for NADPH, whereas deaminating activity was strictly NAD dependent, with apparent Kms of 0.25 (NADPH), 0.15 (NAD+), 1.25 (L-alanine), 0.13 (pyruvate), and 16 (ammonium) mM. The enzyme was inhibited in vitro by pyruvate or L-alanine and had two sulfhydryl groups per subunit which were essential for both aminating and deaminating activities.
Valine dehydrogenase was purified to homogeneity from the crude extracts of Streptomyces aureofaciens. The molecular weight of the native enzyme was 116,000 by equilibrium ultracentrifugation and 118,000 by size exclusion high-performance liquid chromatography. The enzyme was composed of four subunits with molecular weights of 29,000. The isoelectric point was 5.1. The enzyme required NAD+ as a cofactor, which could not be replaced by NADP+. Sulfhydryl reagents inhibited the enzyme activity. The pH optimum was 10.7 for oxidative deamination of L-valine and 9.0 for reductive amination of alpha-ketoisovalerate. The Michaelis constants were 2.5 mM for L-valine and 0.10 mM for NAD+. For reductive amination the Km values were 1.25 mM for alpha-ketoisovalerate, 0.023 mM for NADH, and 18.2 mM for NH4Cl.
A heat-stable L-alanine dehydrogenase was isolated and purified from the extremely thermophilic microorganism, Thermus thermophilus, by affinity chromatography. The enzyme has a molecular weight of 290 000, as determined by the sedimentation equilibrium method, and is composed of six subunits of identical molecular weight as concluded from sodium dodecyl sulphate gel electrophoresis. The enzyme has been characterized in terms of pH- and substrate concentration-dependence of activity, substrate specificity, inhibition by D-alanine and D-cysteine and amino acid composition. The parameters obtained are very similar to those reported for L-alanine dehydrogenase from the mesophilic microorganism, Bacillus subtilis (Yoshida, A. and Freese, E. (1965) Biochim. Biophys. Acta 96, 248--262). The thermal stability of the T. thermophilus enzyme is much greater than that of the B. subtilis enzyme. Activation free energy (delta G), activation enthalpy (delta H) and activation entropy (delta S) values were determined for both the alanine deamination and for the heat inactivation reactions of the thermophilic and mesophilic enzymes. The values obtained for the catalytic reaction were practically equal. However, the two enzymes differed significantly in these parameters determined for the enzyme inactivation, which indicates that the factors ensuring the thermoresistance of the enzyme from T. thermophilus do not affect enzyme activity.
Alcaligenes sp. strain O-1 utilizes three sulphonated aromatic compounds as sole sources of carbon and energy for growth in minimal salts medium-benzenesulphonate (BS), 4-toluenesulphonate (TS) and 2-aminobenzenesulphonate (2AS). The degradative pathway(s) in 2AS-grown cells are initiated with membrane transport, NADH-dependent dioxygenation and meta ring cleavage. The specific activity of the NADH-dependent dioxygenation(s) varied with the growth phase and was maximal near the end of exponential growth for each growth substrate. Cells were harvested at this point from BS-, TS- and 2AS-salts medium. Cells grown with each sulphonated substrate could oxygenate all three compounds, but only 2AS-grown cells consumed 2 mol O2 per mol 2AS or BS or TS. BS- and TS-grown cells consumed 2 mol O2 per mol BS or TS but failed to oxygenate the product of oxygenation of 2AS, 3-sulphocatechol (3SC). These observations were repeated with cell extracts and we concluded that there were two sets of desulphonative pathways in the organism, one for 2AS and one for BS and TS. We confirmed this hypothesis by separating the degradative enzymes from 2AS-, BS- or TS-grown cells. A 2AS dioxygenase system and a 3SC-2,3-dioxygenase (3SC23O) were detected in 2AS-grown cells only. In both BS- and TS-grown cells a dioxygenase system for BS and TS was observed as well as a principal catechol 2,3-dioxygenase (C23O-III), neither of which was present in 2AS-grown cells. The 3SC23O was purified to near homogeneity, found to be monomeric (M(r) 42,000), and to catalyse 2,3-dioxygenation to a product that decayed spontaneously to sulphite and 2-hydroxymuconate. The 2AS dioxygenase system could cause not only deamination of 2AS but also desulphonation of BS and TS. The BS dioxygenase could desulphonate BS and apparently either desulphonate or deaminate 2AS. Strain O-1 thus seems to contain two putative, independently regulated operons involving oxygenation and spontaneous desulphonation(s). One operon encodes at least the 2AS dioxygenase system and 3SC23O whereas the other encodes at least the BS/TS dioxygenase system and C23O-III.
The ski22::Tn917lac insertion mutation in Bacillus subtilis was isolated in a screen for mutations that cause a defect in sporulation but are suppressed by the presence or overexpression of the histidine protein kinase encoded by kinA (spoIIJ). The ski22::Tn917lac insertion mutation was in ald, the gene encoding alanine dehydrogenase. Alanine dehydrogenase catalyzes the deamination of alanine to pyruvate and ammonia and is needed for growth when alanine is the sole carbon or nitrogen source. The sporulation defect caused by null mutations in ald was partly relieved by the addition of pyruvate at a high concentration, indicating that the normal role of alanine dehydrogenase in sporulation might be to generate pyruvate to provide an energy source for sporulation. The spoVN::Tn917 mutation was also found to be an allele of ald. Transcription of ald was induced very early during sporulation and by the addition of exogenous alanine during growth. Expression of ald was normal in all of the regulatory mutants tested, including spo0A, spo0K, comA, sigB, and sigD mutants. The only gene in which mutations affected expression of ald was ald itself. This regulation is probably related to the metabolism of alanine.
Enrichment cultures were prepared under strictly anoxic conditions in medium representing fresh water and containing an organosulfonate as electron donor and carbon source, and nitrate as electron acceptor. The inoculum was from the anaerobic digestor of two communal sewage works. The natural organosulfonates 2-aminoethanesulfonate (taurine), DL-2-amino-3-sulfopropionate (cysteate) and 2-hydroxyethanesulfonate (isethionate) all gave positive enrichments, whereas unsubstituted alkanesulfonates, such as methanesulfonate and arenesulfonates, gave no enrichment. Two representative enrichments were used to obtain pure cultures, and strains NKNTAU (utilizing taurine) and NKNIS (utilizing isethionate) were isolated. Strain NKNTAU was examined in detail. Out of 18 tested organosulfonates, it utilized only one, taurine, and was identified as a novel Alcaligenes sp., a facultatively anaerobic bacterium. Carbon from taurine was converted to cell material and carbon dioxide. The amino group was released as ammonium ion and the sulfonate moiety was recovered as sulfate. Nitrate was reduced to nitrogen gas.
Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding of the biology of this slow-growing pathogen and to help the conception of new prophylactic and therapeutic interventions. The genome comprises 4,411,529 base pairs, contains around 4,000 genes, and has a very high guanine + cytosine content that is reflected in the biased amino-acid content of the proteins. M. tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation.
The structure of the hexameric L-alanine dehydrogenase from Phormidium lapideum reveals that the subunit is constructed from two domains, each having the common dinucleotide binding fold. Despite there being no sequence similarity, the fold of alanine dehydrogenase is closely related to that of the family of D-2-hydroxyacid dehydrogenases, with a similar location of the active site, suggesting that these enzymes are related by divergent evolution. L-alanine dehydrogenase and the 2-hydroxyacid dehydrogenases also use equivalent functional groups to promote substrate recognition and catalysis. However, they are arranged differently on the enzyme surface, which has the effect of directing opposite faces of the keto acid to the dinucleotide in each case, forcing a change in absolute configuration of the product.
Organosulfonates are important natural and man-made compounds, but until recently (T. J. Lie, T. Pitta, E. R. Leadbetter, W. Godchaux III, and J. R. Leadbetter. Arch. Microbiol. 166:204-210, 1996), they were not believed to be dissimilated under anoxic conditions. We also chose to test whether alkane- and arenesulfonates could serve as electron sinks in respiratory metabolism. We generated 60 anoxic enrichment cultures in mineral salts medium which included several potential electron donors and a single organic sulfonate as an electron sink, and we used material from anaerobic digestors in communal sewage works as inocula. None of the four aromatic sulfonates, the three unsubstituted alkanesulfonates, or the N-sulfonate tested gave positive enrichment cultures requiring both the electron donor and electron sink for growth. Nine cultures utilizing the natural products taurine, cysteate, or isethionate were considered positive for growth, and all formed sulfide. Two clearly different pure cultures were examined. Putative Desulfovibrio sp. strain RZACYSA, with lactate as the electron donor, utilized sulfate, aminomethanesulfonate, taurine, isethionate, and cysteate, converting the latter to ammonia, acetate, and sulfide. Strain RZATAU was identified by 16S rDNA analysis as Bilophila wadsworthia. In the presence of, e.g., formate as the electron donor, it utilized, e.g., cysteate and isethionate and converted taurine quantitatively to cell material and products identified as ammonia, acetate, and sulfide. Sulfite and thiosulfate, but not sulfate, were utilized as electron sinks, as was nitrate, when lactate was provided as the electron donor and carbon source. A growth requirement for 1,4-naphthoquinone indicates a menaquinone electron carrier, and the presence of cytochrome c supports the presence of an electron transport chain. Pyruvate-dependent disappearance of taurine from cell extracts, as well as formation of alanine and release of ammonia and acetate, was detected. We suspected that sulfite is an intermediate, and we detected desulfoviridin (sulfite reductase). We thus believe that sulfonate reduction is one aspect of a respiratory system transferring electrons from, e.g., formate to sulfite reductase via an electron transport system which presumably generates a proton gradient across the cell membrane.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Alanine dehydrogenase was purified to near homogeneity from cell-free extract of Streptomyces aureofaciens, which produces tetracycline. The molecular weight of the enzyme determined by size-exclusion high-performance liquid chromatography was 395 000. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis was 48 000, indicating that the enzyme consists of eight subunits with similar molecular weight. The isoelectric point of alanine dehydrogenase is 6.7. The pH optimum is 10.0 for oxidative deamination of L-alanine and 8.5 for reductive amination of pyruvate. K
M values were 5.0 mM for L-alanine and 0.11 mM for NAD+. K
M values for reductive amination were 0.56 mM for pyruvate, 0.029 mM for NADH and 6.67 mM for NH4Cl.
The degradation of l-alanine by three strains of sulfate-reducing bacteria that can grow with l-alanine as an energy source was investigated. In Desulfotomaculum ruminis and most likely also in two marine Desulfovibrio strains alanine is converted to pyruvate via an NAD-dependent alanine dehydrogenase. D. ruminis contained high activities of soluble NADH and NADPH dehydrogenases. In the marine strains the activities were much lower and the NADH dehydrogenase was partly associated with the membrane fraction.
Organosulfonates are important natural and man-made compounds, but until recently (T. J. Lie, T. Pitta, E. R. Leadbetter, W. Godchaux III, and J. R. Leadbetter. Arch. Microbiol. 166:204-210, 1996), they were not believed to be dissimilated under anoxic conditions. We also chose to test whether alkane- and arenesulfonates could serve as electron sinks in respiratory metabolism. We generated 60 anoxic enrichment cultures in mineral salts medium which included several potential electron donors and a single organic sulfonate as an electron sink, and we used material from anaerobic digestors in communal sewage works as inocula. None of the four aromatic sulfonates, the three unsubstituted alkanesulfonates, or the N-sulfonate tested gave positive enrichment cultures requiring both the electron donor and electron sink for growth. Nine cultures utilizing the natural products taurine, cysteate, or isethionate were considered positive for growth, and all formed sulfide. Two clearly different pure cultures were examined. Putative Desulfovibrio sp. strain RZACYSA, with lactate as the electron donor, utilized sulfate, aminomethanesulfonate, taurine, isethionate, and cysteate, converting the latter to ammonia, acetate, and sulfide. Strain RZATAU was identified by 16S rDNA analysis as Bilophila wadsworthia. In the presence of, e.g., formate as the electron donor, it utilized, e.g., cysteate and isethionate and converted taurine quantitatively to cell material and products identified as ammonia, acetate, and sulfide. Sulfite and thiosulfate, but not sulfate, were utilized as electron sinks, as was nitrate, when lactate was provided as the electron donor and carbon source. A growth requirement for 1,4- naphthoquinone indicates a menaquinone electron carrier, and the presence of cytochrome c supports the presence of an electron transport chain. Pyruvate-dependent disappearance of taurine from cell extracts, as well as formation of alanine and release of ammonia and acetate, was detected. We suspected that sulfite is an intermediate, and we detected desulfoviridin (sulfite reductase). We thus believe that sulfonate reduction is one aspect of a respiratory system transferring electrons from, e.g., formate to sulfite reductase via an electron transport system which presumably generates a proton gradient across the cell membrane.
Metazachlor (R-CH2-Cl), a chloroacetanilide herbicide, is converted in soil to products including the ethanesulfonate metabolite (R-CH2-SO3¯; BH 479-8). Nothing is known about the degradation of the ethanesulfonates of this class of herbicides. We used inocula derived from five sources for enrichment cultures to utilize R-CH2-SO3¯ as a sole sulfur source for the growth of microorganisms. Each culture yielded bacteria that caused the disappearance of R-CH2-SO3¯ and the formation of a product identified as the glycolate metabolite (R-CH2-OH; BH 479-1) by mass spectrometry. A pure culture, strain HL1, was isolated, and this bacterium quantitatively desulfonated R-CH2-SO3¯, the sulfur being recovered in cell protein. Recovery of the organic moiety was usually about 80%. A second ethanesulfonate (R-CH2-SO3¯) and two alkylsulfonates, but not taurine, were utilized by strain HL1 as sulfur sources.
The l-alanine dehydrogenase (ADH) of Anabaena cylindrica has been purified 700-fold. It has a molecular weight of approximately 270000, has 6 sub-units, each of molecular weight approximately 43000, and shows activity both in the aminating and deaminating directions. The enzyme is NADH/NAD+ specific and oxaloacetate can partially substitute for pyruvate. The K
m
app
for NAD+ is 14 μM and 60 μM at low and high NAD+ concentrations, respectively. The K
m
app
for l-alanine is 0.4 mM, that for pyruvate is 0.11 mM, and that for oxaloacetate is 3.0 mM. The K
m
app
for NH
4+varies from 8–133 mM depending on the pH, being lowest at high pH levels (pH 8.7 or above). Alanine, serine and glycine inhibit ADH activity in the aminating direction. The enzyme is active both in heterocysts and vegetative cells and activity is higher in nitrogen-starved cultures than in N2-fixing cultures. The data suggest that although alanine is formed by the aminating activity of ADH, entry of newly fixed ammonia into organic combination does not occur primarily via ADH in N2-fixing cultures of A. cylindrica. Ammonia assimilation via ADH may be important in cultures with an excess of available nitrogen. The deaminating activity of the enzyme may be important under conditions of nitrogen-deficiency.
1. The bacterial distribution of alanine dehydrogenase (L-alanine:NAD+ oxidoreductase, deaminating, EC 1.4.1.1) was investigated, and high activity was found in Bacillus species. The enzyme has been purified to homogeneity and crystallized from B. sphaericus (IFO 3525), in which the highest activity occurs. 2. The enzyme has a molecular weight of about 230 000, and is composed of six identical subunits (Mr 38 000). 3. The enzyme acts almost specifically on L-alanine, but shows low amino-acceptor specificity; pyruvate and 2-oxobutyrate are the most preferable substrates, and 2-oxovalerate is also animated. The enzyme requires NAD+ as a cofactor, which cannot be replaced by NADP+. 4. The enzyme is stable over a wide pH range (pH 6.0--10.0), and shows maximum reactivity at approximately pH 10.5 and 9.0 for the deamination and amination reactions, respectively. 5. Alanine dehydrogenase is inhibited significantly by HgCl2, p-chloromercuribenzoate and other metals, but none of purine and pyrimidine bases, nucleosides, nucleotides, flavine compounds and pyridoxal 5'-phosphate influence the activity. 6. The reductive amination proceeds through a sequential ordered ternary-binary mechanism. NADH binds first to the enzyme followed by ammonia and pyruvate, and the products are released in the order of L-ALANINE AND NAD+. The Michaelis constants are as follows: NADH (10 microM), ammonia (28.2 mM), pyruvate (1.7 mM), L-alanine (18.9 mM) and NAD+ (0.23 mM). 7. The pro-R hydrogen at C-4 of the reduced nicotinamide ring of NADH is exclusively transferred to pyruvate; the enzyme is A-stereospecific.
The gene encoding alanine dehydrogenase (EC 1.4.1.1) from a mesophile, Bacillus sphaericus, was cloned, and its complete DNA sequence was determined. In addition, the same gene from a moderate thermophile, B. stearothermophilus, was analyzed in a similar manner. Large parts of the two translated amino acid sequences were confirmed by automated Edman degradation of tryptic peptide fragments. Each alanine dehydrogenase gene consists of a 1116-bp open reading frame and encodes 372 amino acid residues corresponding to the subunit (Mr = 39,500-40,000) of the hexameric enzyme. The similarity of amino acid sequence between the two alanine dehydrogenases with distinct thermostabilities is very high (greater than 70%). The nonidentical residues are clustered in a few regions with relatively short length, which may correlate with the difference in thermal stability of the enzymes. Homology search of the primary structures of both alanine dehydrogenases with those of other pyridine nucleotide-dependent oxidoreductases revealed significant sequence similarity in the regions containing the coenzyme binding domain. Interestingly, several catalytically important residues in lactate and malate dehydrogenases are conserved in the primary structure of alanine dehydrogenases at matched positions with similar mutual distances.
Consensus sequence patterns for beta-alpha-beta folds binding FAD, NAD and GTP were constructed on the basis of 11 steric and physicochemical properties. These property patterns permit detection and distinction of the respective nucleotide-binding sites on the basis of amino acid sequence analysis alone. The SWISS-PROT database (release 9) was screened with the three calculated patterns, and nucleotide-binding sites identified are presented. They correspond to existing structure data (if known). For the detected sequence segments we are able to predict the beta-alpha-beta motif as well as the respective binding sites. For some of the proteins so detected a nucleotide-binding capacity has not previously been reported.
An improved procedure for staining of proteins following separation in polyacrylamide gels is described which utilizes the colloidal properties of Coomassie Brilliant Blue G-250 and R-250. The new method is based on addition of 20% v/v methanol and higher concentrations of ammonium sulfate to the staining solution previously described. The method combines the advantage of much shorter staining time with high sensitivity, a clear background not requiring destaining, stepwise staining, and stable fixation after staining. The method has been applied to staining of polyacrylamide gels after sodium dodecyl sulfate-electrophoresis and isoelectric focusing in carrier ampholyte-generated pH gradients.
Alanine dehydrogenase was purified to homogeneity from a cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1180-fold to give a 21% yield, using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The relative molecular mass of the native enzyme was determined to be 210,000 or 205,000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of Mr 51,000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The Km were 10.0 mM for L-alanine and 0.18 mM for NAD+. Km values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH4+ and 0.05 mM for NADH. Oxidative deamination of L-alanine proceeds through a sequential-ordered binary-ternary mechanism in which NAD+ binds first to the enzyme, followed by alanine, and products are released in the order ammonia, pyruvate and NADH.
An amino acid sequence "fingerprint" has been derived that can be used to test if a particular sequence will fold into a beta alpha beta-unit with ADP-binding properties. It was deduced from a careful analysis of the known three-dimensional structures of ADP-binding beta alpha beta-folds. This fingerprint is in fact a set of 11 rules describing the type of amino acid that should occur at a specific position in a peptide fragment. The total length of this fingerprint varies between 29 and 31 residues. By checking against all possible sequences in a database, it appeared that every peptide, which exactly follows this fingerprint, does indeed fold into an ADP-binding beta alpha beta-unit.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
This review is an exhaustive description of the biochemistry and enzymology of all 17 known NAD(P)(+)-amino acid dehydrogenases. These enzymes catalyze the oxidative deamination of an amino acid to its keto acid and ammonia, with the concomitant reduction of either NAD+ or NADP+. These enzymes have many important applications in industrial and medical settings and have been the object of prodigious enzymological research. This article describes all that is known about the poorly characterized members of the family and contains detailed information on the better characterized enzymes, including valine, phenylalanine, leucine, alanine, and glutamate dehydrogenases. The latter three enzymes have been the subject of extensive enzymological experimentation, and, consequently, their chemical mechanisms are discussed. The three-dimensional structure of the Clostridium symbiosum glutamate dehydrogenase has been determined recently and remains the only structure known of any amino acid dehydrogenase. The three-dimensional structure and its implications to the chemical mechanisms and rate-limiting steps of the amino acid dehydrogenase family are discussed.
Alanine dehydrogenase (AlaDH) was purified to homogeneity from cell-free extracts of a non-N2-fixing filamentous cyanobacterium, Phormidium lapideum. The molecular mass of the native enzyme was 240 kDa, and SDS-PAGE revealed a minimum molecular mass of 41 kDa, suggesting a six-subunit structure. The NH2 terminal amino acid residues of the purified AlaDH revealed marked similarity with that of other AlaDHs. The enzyme was highly specific for L-alanine and NAD+, but showed relatively low amino-acceptor specificity. The pH optimum was 8.4 for reductive amination of pyruvate and 9.2 for oxidative deamination of L-alanine. The Km values were 5.0 mM for L-alanine and 0.04 mM for NAD+, 0.33 mM for pyruvate, 60.6 mM for NH4+ (pH 8.7), and 0.02 mM for NADH. Various L-amino acids including alanine, serine, threonine, and aromatic amino acids, inhibited the aminating reaction. The enzyme was inactivated upon incubation with pyridoxal 5'-phosphate (PLP) followed by reduction with sodium borohydride. The copresence of NADH and pyruvate largely protected the enzyme against the inactivation by PLP.
Alanine dehydrogenase [EC 1. 4. 1. 1] was purified to homogeneity from a crude extract of Enterobacter aerogenes ICR 0220. The enzyme had a molecular mass of about 245 kDa and consisted of six identical subunits. The enzyme showed maximal activity at about pH 10.9 for the deamination of L-alanine and at about pH 8.7 for the amination of pyruvate. The enzyme required NAD+ as a coenzyme. Analogs of NAD+, deamino-NAD+ and nicotinamide guanine dinucleotide served as coenzymes. Initial-velocity and product inhibition studies suggested that the deamination of L-alanine proceeded through a sequential ordered binary-ternary mechanism. NAD+ bound first to the enzyme, followed by L-alanine, and the products were released in the order of ammonia, pyruvate, and NADH. The Km were 0.47 mM for L-alanine, 0.16 mM for NAD+, 0.22 mM for pyruvate, 0.067 mM for NADH, and 66.7 mM for ammonia. The Km for L-alanine was the smallest in the alanine dehydrogenases studied so far. The enzyme gene was cloned into Escherichia coli JM109 cells and the nucleotides were sequenced. The deduced amino acid sequence was very similar to that of the alanine dehydrogenase from Bacillus subtilis. However, the Enterobacter enzyme has no cysteine residue. In this respect, the Enterobacter enzyme is different from other alanine dehydrogenases.
An NAD-dependent, morpholine-stimulated l-alanine dehydrogenase activity was detected in crude extracts from morpholine-, pyrrolidine-, and piperidine-grown cells of Mycobacterium strain HE5. Addition of morpholine to the assay mixture resulted in an up to 4.6-fold increase of l-alanine dehydrogenase activity when l-alanine was supplied at suboptimal concentration. l-Alanine dehydrogenase was purified to near homogeneity using a four-step purification procedure. The native enzyme had a molecular mass of 160 kDa and contained one type of subunit with a molecular mass of 41 kDa, indicating a tetrameric structure. The sequence of 30 N-terminal amino acids was determined and showed a similarity of up to 81% to that of various alanine dehydrogenases. The pH optimum for the oxidative deamination of l-alanine, the only amino acid converted by the enzyme, was determined to be pH 10.1, and apparent K
m values for l-alanine and NAD were 1.0 and 0.2 mM, respectively. K
m values of 0.6, 0.02, and 72 mM for pyruvate, NADH, and NH4+, respectively, were estimated at pH 8.7 for the reductive amination reaction.
Incubation of marine sediment in anoxic, sulphate-rich medium in the presence of naphthalene resulted in the enrichment of sulphate-reducing bacteria. Pure cultures with short, oval cells (1.3 by 1.3-1.9 microm) were isolated that grew with naphthalene as the only organic carbon source and electron donor for sulphate reduction to sulphide. One strain, NaphS2, was characterized. It affiliated with completely oxidizing sulphate-reducing bacteria of the delta-subclass of the Proteobacteria, as revealed by 16S rRNA sequence analysis. 2-Naphthoate, benzoate, pyruvate and acetate were used in addition to naphthalene. Quantification of substrate consumption, sulphide formation and formed cell mass revealed that naphthalene was completely oxidized with sulphate as the electron acceptor.
Enzymic properties of alanine dehydrogenase (l-alanine:NAD oxidoreductase, EC 1.4.1 1) from Bacillus subtilis were examined using a highly purified crystalline enzyme preparation. 1. 1. Optimal pH for the amination reaction was 8.8-9.0, whereas it was 10-10.5 for the deamination reaction. 2. 2. Michaelis constants for the primary substrates were determined. The equilibrium constant (Ka) of the reaction was 3.1·10-14. 3. 3. Analogs of l-alanine and pyruvate that were accepted as substrates were in order of decreasing activity, for amination: l-α-aminobutyrate, l-valine, 1-isoleucine. l-serine and l-norvaline; for deamination: α-ketoglutarate, glyoxalate, hydroxypyruvate, α-ketovalerate, α-ketoisovalerate, α-ketoisocaproate and α-ketocaproate. 4. 4. The enzyme was partially inactivated by dilution, without significant change of molecular weight. 5. 5. Divalent metal ions and p-chloromercuribenzoate inactivated the enzyme, mercuric ion being most effective, the inactivation was resersed by cysteine.
Although comprising less than 0.01% of the normal human gastrointestinal microbiota, Bilophila wadsworthia is the third most common anaerobe recovered from clinical material obtained from patients with perforated and gangrenous appendicitis. Since its discovery in 1988, B. wadsworthia has been recovered from clinical specimens associated with a variety of infections, including sepsis, liver abscesses, cholecystitis, Fournier's gangrene, soft tissue abscesses, empyema, osteomyelitis, Bartholinitis, and hidradenitis suppurativa. In addition, it has been found in the saliva and vaginal fluids of asymptomatic adults and even in the periodontal pockets of dogs. The organism is a saccharolytic, fastidious, and is easily recognized by its strong catalase reaction with 15% H2O2, production of hydrogen sulfide, and growth stimulation by bile (oxgall) and pyruvate. Approximately 75% of strains are urease positive. When grown on pyruvate-containing media, > 85% of strains demonstrate beta-lactamase production. Ribosomal RNA-based phylogenetic studies show Bilophila to be a homogeneous species, most closely related to Desulfovibrio species. Both adherence to human cells and endotoxin have been observed, and preliminary work suggests that environmental iron has a role in expression of outer membrane proteins. Penicillin-binding proteins appear to mediate the organism's susceptibility to at least some beta-lactam agents, which induce spheroplast formation that results in a haze of growth on agar dilution susceptibility test plates which is difficult to interpret. Bilophilastrains are inhibited in vitro by most antibiotics.
Alanine dehydrogenase of the N 2 -fixing blue-green alga, Anabaena cylindrica
- P Stewart
- Wd
P, Stewart WD (1975) Alanine dehydrogenase of the N 2 -fixing blue-green alga, Anabaena cylindrica. Arch Microbiol 107:115–124
Methods of enzymatic analysis
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Bergmeyer HU (1983) Methods of enzymatic analysis, 3rd edn.
Verlag Chemie, Weinheim
Current protocols in molecular biology Analysis of the structure and substrate binding of Phormidium lapideum alanine dehydrogenase
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Smith JA, Struhl K (eds) (1987) Current protocols in molecular
biology. Wiley, New York
Baker PJ, Sawa Y, Shibata H, Sedelnikova SE, Rice DW (1998)
Analysis of the structure and substrate binding of Phormidium
lapideum alanine dehydrogenase. Nat Struct Biol 5:561–567