[Show abstract][Hide abstract] ABSTRACT: The substrate reactivity of the flavoenzyme 2-hydroxybiphenyl 3-monooxygenase (EC 18.104.22.168, HbpA) was changed by directed evolution using error-prone PCR. In situ screening of mutant libraries resulted in the identification of proteins with increased activity towards 2-tert-butylphenol and guaiacol (2-methoxyphenol). One enzyme variant contained amino acid substitutions V368A/L417F, which were inserted by two rounds of mutagenesis. The double replacement improved the efficiency of substrate hydroxylation by reducing the uncoupled oxidation of NADH. With guaiacol as substrate, the two substitutions increased Vmax from 0.22 to 0.43 units mg1 protein and decreased the K'm from 588 to 143 ?M, improving k'cat/K'm by a factor of 8.2. With 2-tert-butylphenol as the substrate, k'cat was increased more than 5-fold. Another selected enzyme variant contained amino acid substitution I244V and had a 30␑igher specific activity with 2-sec-butylphenol, guaiacol, and the "natural" substrate 2-hydroxybiphenyl. The K'm for guaiacol decreased with this mutant, but the K'm for 2-hydroxybiphenyl increased. The primary structure of HbpA shares 20.1␜equence identity with phenol 2-monooxygenase from Trichosporon cutaneum. Structure homology modeling with this three-domain enzyme suggests that Ile244 of HbpA is located in the substrate binding pocket and is involved in accommodating the phenyl substituent of the phenol. In contrast, Val368 and Leu417 are not close to the active site and would not have been obvious candidates for modification by rational design.
[Show abstract][Hide abstract] ABSTRACT: The biotransformation of four different classes of aromatic compounds by the Escherichia coli strain DH5α(pTCB 144), which contained the chlorobenzene dioxygenase (CDO) fromPseudomonas sp. strain P51, was examined. CDO oxidized biphenyl as well as monochlorobiphenyls to the correspondingcis-2,3-dihydro-2,3-dihydroxy derivatives, whereby oxidation occurred on the unsubstituted ring. No higher substituted biphenyls
were oxidized. The absolute configurations of several monosubstituted cis-benzene dihydrodiols formed by CDO were determined. All had an S configuration at the carbon atom in meta position to the substituent on the benzene nucleus. With one exception, the enantiomeric excess of several 1,4-disubstituted
cis-benzene dihydrodiols formed by CDO was higher than that of the products formed by two toluene dioxygenases. Naphthalene was
oxidized to enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. All absolute configurations were identical to those of the products formed by toluene
dioxygenases of Pseudomonas putida UV4 and P. putida F39/D. The formation rate of (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene was significantly higher (about 45 to 200%) than those of several monosubstituted cis-benzene dihydrodiols and more than four times higher than the formation rate of cis-benzene dihydrodiol. A new gas chromatographic method was developed to determine the enantiomeric excess of the oxidation
[Show abstract][Hide abstract] ABSTRACT: Pseudomonas azelaica HBP1 degrades the toxic substance 2-hydroxybiphenyl (2-HBP) by means of three enzymes that are encoded by structural genes
hbpC, hbpA, andhbpD. These three genes form a small noncontiguous cluster. Their expression is activated by the product of regulatory genehbpR, which is located directly upstream of thehbpCAD genes. The HbpR protein is a transcription activator and belongs to the so-called XylR/DmpR subclass within the NtrC family
of transcriptional activators. Transcriptional fusions between the different hbp intergenic regions and the luxABgenes of Vibrio harveyi in P. azelaica and inEscherichia coli revealed the existence of two HbpR-regulated promoters; one is located in front of hbpC, and the other one is located in front of hbpD. Northern analysis confirmed that the hbpC and hbpA genes are cotranscribed, whereas the hbpD gene is transcribed separately. No transcripts comprising the entire hbpCADcluster were detected, indicating that transcription from PhbpC is terminated after the hbpAgene. E. coli mutant strains lacking the structural genes for the RNA polymerase ς54 subunit or for the integration host factor failed to express bioluminescence from PhbpC- and PhbpD
-luxABfusions when a functional hbpR gene was provided intrans. This pointed to the active role of ς54and integration host factor in transcriptional activation from these promoters. Primer extension analysis revealed that both
PhbpC and PhbpD contain the typical motifs at position −24 (GG) and −12 (GC) found in ς54-dependent promoters. Analysis of changes in the synthesis of the hbp mRNAs, in activities of the 2-HBP pathway enzymes, and in concentrations of 2-HBP intermediates during the first 4 h after
induction of continuously grown P. azelaica cells with 2-HBP demonstrated that the specific transcriptional organization of the hbp genes ensured smooth pathway expression.
Full-text · Article · Feb 2001 · Journal of Bacteriology
[Show abstract][Hide abstract] ABSTRACT: cis-Chlorobenzene dihydrodiol dehydrogenase (CDD) from Pseudomonas sp. strain P51, cloned into Escherichia coli DH5alpha(pTCB149) was able to oxidize cis-dihydrodihydroxy derivatives (cis-dihydrodiols) of dihydronaphthalene, indene, and four para-substituted toluenes to the corresponding catechols. During the incubation of a nonracemic mixture of cis-1,2-indandiol, only the (+)-cis-(1R,2S) enantiomer was oxidized; the (-)-cis-(S,2R) enantiomer remained unchanged. CDD oxidized both enantiomers of cis-1,2-dihydroxy-1,2,3, 4-tetrahydronaphthalene, but oxidation of the (+)-cis-(1S,2R) enantiomer was delayed until the (-)-cis-(1R,2S) enantiomer was completely depleted. When incubated with nonracemic mixtures of para-substituted cis-toluene dihydrodiols, CDD always oxidized the major enantiomer at a higher rate than the minor enantiomer. When incubated with racemic 1-indanol, CDD enantioselectively transformed the (+)-(1S) enantiomer to 1-indanone. This stereoselective transformation shows that CDD also acted as an alcohol dehydrogenase. Additionally, CDD was able to oxidize (+)-cis-(1R,2S)-dihydroxy-1, 2-dihydronaphthalene, (+)-cis-monochlorobiphenyl dihydrodiols, and (+)-cis-toluene dihydrodiol to the corresponding catechols.
Full-text · Article · Jan 2000 · Applied and Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: Chirality is a geometric attribute; objects or molecules for which the originals and their mirror images cannot be superposed are chiral, whereas objects that can be superposed on their mirror images are achiral. Commonplace examples of chiral objects are a person’s right and left hand (Fig. 1 A), clockwise and counterclockwise threaded screws, and seashells. An alternative definition of chirality is based on symmetry elements; a chiral object lacks reflectional symmetry (Sheldon, 1993). The most common type of chiral molecule contains a tetrahedral carbon atom attached to four different groups; the carbon atom is the stereogenic center of the molecule. The two possible structures of such compounds behave like image and mirror image that are not superimposable on each other. Such stereoisomers are called enantiomers (Greek: enantios, opposite). Lactic acid, for example, is a chiral molecule and exists as a pair of enantiomers (Fig. IB). If a molecule possesses more than one stereogenic center, for example, n
asymmetric carbon atoms, then 2n
stereoisomers are theoretically possible. In practice, sometimes less than 2n
isomers exist because of a plane of symmetry or a rigid structure (see tartaric acid, Fig. 1C, D).
No preview · Article · Jan 2000 · Advances in microbial ecology
[Show abstract][Hide abstract] ABSTRACT: 2-Hydroxybiphenyl 3-monooxygenase (EC 22.214.171.124) from Pseudomonas azelaica HBP1 is an FAD-dependent aromatic hydroxylase that catalyzes the conversion of 2-hydroxybiphenyl to 2, 3-dihydroxybiphenyl in the presence of NADH and oxygen. The catalytic mechanism of this three-substrate reaction was investigated at 7 degrees C by stopped-flow absorption spectroscopy. Various individual steps associated with catalysis were readily observed at pH 7.5, the optimum pH for enzyme turnover. Anaerobic reduction of the free enzyme by NADH is a biphasic process, most likely reflecting the presence of two distinct enzyme forms. Binding of 2-hydroxybiphenyl stimulated the rate of enzyme reduction by NADH by 2 orders of magnitude. The anaerobic reduction of the enzyme-substrate complex involved the formation of a transient charge-transfer complex between the reduced flavin and NAD(+). A similar transient intermediate was formed when the enzyme was complexed with the substrate analog 2-sec-butylphenol or with the non-substrate effector 2,3-dihydroxybiphenyl. Excess NAD(+) strongly stabilized the charge-transfer complexes but did not give rise to the appearance of any intermediate during the reduction of uncomplexed enzyme. Free reduced 2-hydroxybiphenyl 3-monooxygenase reacted rapidly with oxygen to form oxidized enzyme with no appearance of intermediates during this reaction. In the presence of 2-hydroxybiphenyl, two consecutive spectral intermediates were observed which were assigned to the flavin C(4a)-hydroperoxide and the flavin C(4a)-hydroxide, respectively. No oxygenated flavin intermediates were observed when the enzyme was in complex with 2, 3-dihydroxybiphenyl. Monovalent anions retarded the dehydration of the flavin C(4a)-hydroxide without stabilization of additional intermediates. The kinetic data for 2-hydroxybiphenyl 3-monooxygenase are consistent with a ternary complex mechanism in which the aromatic substrate has strict control in both the reductive and oxidative half-reaction in a way that reactions leading to substrate hydroxylation are favored over those leading to the futile formation of hydrogen peroxide. NAD(+) release from the reduced enzyme-substrate complex is the slowest step in catalysis.
Full-text · Article · Dec 1999 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Sphingomonas herbicidovorans MH was isolated from a dichlorprop-degrading soil column. It is able to grow on phenoxyalkanoic acid herbicides, such as mecoprop, dichlorprop, 2,4-D, MCPA, and 2,4-DB. Strain MH utilizes both enantiomers of the chiral herbicides mecoprop and dichlorprop as sole carbon and energy sources. Enantiomer-specific uptake systems are responsible for transporting the acidic substrates across the cell membrane. Catabolism is initiated by two enantiomer-specific alpha-ketoglutarate-dependent dioxygenases that catalyze the cleavage of the ether bond of the respective enantiomer to yield the corresponding phenol and pyruvate. Therefore selective degradation of the enantiomers of mecoprop and dichlorprop by strain MH is not only due to enantioselective catabolism but also to enantioselective transport.
No preview · Article · Nov 1999 · Journal of Industrial Microbiology
[Show abstract][Hide abstract] ABSTRACT: We describe the biocatalytic production of 3-phenylcatechol from 2-phenylphenol with the whole cell biocatalyst Escherichia coli JM101 (pHBP461). The recombinant produces 2-hydroxybiphenyl 3-monooxygenase, an enzyme from Pseudomonas azelaica HBP1. This enzyme introduces a hydroxyl-group at the C3-position of a variety of 2-substituted phenols, such as 2-phenylphenol. This permits the biocatalytic production of 3-substituted catechols, which are difficult to synthesize chemically. Both 2-phenylphenol and 3-phenylcatechol are highly toxic to E. coli. The toxic effects of 2-phenylphenol were minimized by feeding this substrate to the reactor at a rate slightly below the maximum biooxidation rate. As a result, the substrate concentration in the reactor remained below toxic levels during the bioconversion. The toxic product formed was removed by continuous adsorption on the solid resin Amberlite XAD-4. To this end the reaction mixture, containing the biocatalyst, was pumped continuously through an external loop with a fluidized bed of the resin. This resin efficiently and quantitatively adsorbed both 3-phenylcatechol and the remaining trace amounts of 2-phenylphenol. Consequently, the concentrations of these compounds were kept at subtoxic levels (below 100 mg L-1) and gram amounts of 3-phenylcatechol were produced with space-time yields of up to 0.39 g L-1 h-1. The product was recovered from the resin by acidic methanol elution and purified by recrystallization from n-hexane resulting in overall yields exceeding 59%. The optimized system served as a surprisingly simple and efficient integrated process, that allows the bioconversion of toxic substrates to toxic products with whole cell biocatalysts.
No preview · Article · Apr 1999 · Biotechnology and Bioengineering
[Show abstract][Hide abstract] ABSTRACT: Aerobic degradation experiments with the racemic mixtures of mecoprop and dichlorprop revealed that activated sludge collected from the aeration tank of a municipal waste water treatment plant degraded both enantiomers of mecoprop and dichlorprop within 7 days, albeit in an enantioselective manner; the (S) enantiomers were preferentially degraded. Mecoprop, dichlorprop, and 2,4-D were completely metabolized under aerobic conditions, as shown by the 86-98% elimination of dissolved organic carbon. Under anaerobic conditions, the concentration of 2,4-D decreased exponentially with a first-order reaction rate constant of 0.24 per day and without a lag-phase. After an incubation time of 17 days, 2,4-D was completely removed. 2,4-Dichlorophenol was the main metabolite of anaerobic 2,4-D degradation; only traces of 4-chlorophenol were detected. In contrast, the chiral phenoxypropionic acid herbicides mecoprop and dichlorprop persisted under anaerobic conditions during 49 days of incubation.
[Show abstract][Hide abstract] ABSTRACT: Sphingomonas herbicidovorans MH was able to completely degrade both enantiomers of the chiral herbicide dichlorprop [(RS)-2-(2,4-dichlorophenoxy)propanoic acid], with preferential degradation of the (S) enantiomer over the (R) enantiomer. These results are in agreement with the recently reported enantioselective degradation of mecoprop [(RS)-2-(4-chloro-2-methylphenoxy)propanoic acid] by this bacterium (C. Zipper, K. Nickel, W. Angst, and H.-P. E. Kohler, Appl. Environ. Microbiol. 62:4318-4322, 1996). Uptake of (R)-dichlorprop, (S)-dichlorporp, and 2,4-D (2,4-dichlorophenoxyacetic acid) was inducible. Initial uptake rates of cells grown on the respective substrate showed substrate saturation kinetics with apparent affinity constants (Kt) of 108, 93, and 117 microM and maximal velocities (Vmax) of 19, 10, and 21 nmol min-1 mg of protein-1 for (R)-dichlorprop, (S)-dichlorprop, and 2,4-D, respectively. Transport of (R)-dichlorprop, (S)-dichlorprop, and 2,4-D was completely inhibited by various uncouplers and by nigericin but was only marginally inhibited by valinomycin and by the ATPase inhibitor N,N'-dicyclohexylcarbodiimine. Experiments on the substrate specificity of the putative transport systems revealed that (R)-dichlorprop uptake was inhibited by (R)-mecoprop but not by (S)-mecoprop, (S)-dichlorprop, or 2,4-D. On the other hand, the (S)-dichlorprop transport was inhibited by (S)-mecoprop but not by (R)-mecoprop, (R)-dichlorprop, or 2,4-D. These results provide evidence that the first step in the degradation of dichlorprop, mecoprop, and 2,4-D by S. herbicidovorans is active transport and that three inducible, proton gradient-driven uptake systems exist: one for (R)-dichlorprop and (R)-mecoprop, another for (S)-dichlorprop and (S)-mecoprop, and a third for 2,4-D.
Preview · Article · Aug 1998 · Journal of Bacteriology
[Show abstract][Hide abstract] ABSTRACT: High-performance capillary electrophoresis (HPCE) with α-cyclodextrin as the chiral selector was applied to separate the enantiomers of p-sulfophenyl-2-butyrate (SP2B) and p-sulfophenyl-3-butyrate (SP3B), which occur as biodegradation intermediates of linear alkylbenzenesulfonates (LAS), the widely used anionic surfactants. With this analytical method, we studied the transformation of both SP3B enantiomers in a laboratory batch incubation with activated sewage sludge of a municipal wastewater treatment plant. (S)-(+)-SP3B and (R)-(-)-SP3B could be detected in mechanically treated sewage effluent. After enrichment on graphitized carbon black (Carbopack B), the extracts were analyzed by HPLC with UV diode array and fluorescence detection as well as by HPCE with UV diode array detection. Quantification of SP3B in a 24-h composite sample of primary sewage effluent yielded 34 μg/L (limit of detection, 0.1 μg/L) of the racemic mixture determined by HPLC and 18 μg/L of each enantiomer measured by HPCE (limit of detection, 1 μg/L).
No preview · Article · Mar 1998 · Analytical Chemistry
[Show abstract][Hide abstract] ABSTRACT: Cell extracts of Sphingomonas herbicidovorans MH grown on (R)-mecoprop contained an enzyme activity that selectively converted (R)-mecoprop to 4-chloro-2-methylphenol, whereas extracts of cells grown on (S)-mecoprop contained an enzyme activity selective for the S enantiomer. Both reactions were dependent on alpha-ketoglutarate and ferrous ions. Besides 4-chloro-2-methylphenol, pyruvate and succinate were detected as products of the reactions. Labeling experiments with (18)O2 revealed that both enzyme activities catalyzed a dioxygenation reaction. One of the oxygen atoms of pyruvate and one of the oxygen atoms of succinate were derived from molecular oxygen. Analysis of cell extracts obtained from cells grown on different substrates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that growth on (R)-mecoprop and (S)-mecoprop caused the appearance of prominent protein bands at 34 and 32 kDa, respectively. Both protein bands were present when cells grew on the racemic mixture. The results demonstrate that S. herbicidovorans initiated the degradation of each enantiomer of mecoprop by a specific alpha-ketoglutarate-dependent dioxygenase. By comparing conversion rates of various phenoxy herbicides, we confirmed that the two enzyme activities were distinct from that of TfdA, which catalyzes the first step in the degradation of 2,4-dichlorophenoxyacetic acid in Ralstonia eutropha JMP134.
Full-text · Article · Dec 1997 · Journal of Bacteriology
[Show abstract][Hide abstract] ABSTRACT: 2-Hydroxybiphenyl 3-monooxygenase (HbpA), the first enzyme of 2-hydroxybiphenyl degradation inPseudomonas azelaica HBP1, was purified 26-fold with a yield of 8% from strain HBP1 grown on 2-hydroxybiphenyl. The enzyme was also purified from
a recombinant of Escherichia coliJM109, which efficiently expressed the hbpA gene. Computer densitometry of scanned slab gels revealed a purity of over 99% for both enzyme preparations. Gel filtration,
subunit cross-linking, and SDS-polyacrylamide gel electrophoresis showed that the enzyme was a homotetramer with a molecular
mass of 256 kDa. Each subunit had a molecular mass of 60 kDa containing one molecule of noncovalently bound FAD. The monooxygenase
had a pI of 6.3. It catalyzed the NADH-dependent ortho-hydroxylation of 2-hydroxybiphenyl to 2,3-dihydroxybiphenyl. Molecular oxygen was the source of the additional oxygen of
the product. The enzyme hydroxylated various phenols with a hydrophobic side chain adjacent to the hydroxy group. All substrates
effected partial uncoupling of NADH oxidation from hydroxylation with the concomitant formation of hydrogen peroxide. 2,3-Dihydroxybiphenyl,
the product of the reaction with 2-hydroxybiphenyl, was a non-substrate effector that strongly facilitated NADH oxidation
and hydrogen peroxide formation without being hydroxylated and also was an inhibitor. The apparentK
m values (30 °C, pH 7.5) were 2.8 μm for 2-hydroxybiphenyl, 26.8 μm for NADH, and 29.2 μm for oxygen. The enzyme was inactivated byp-hydroxymercuribenzoate, a cysteine-blocking reagent. In the presence of 2-hydroxybiphenyl, the enzyme was partly protected
against the inactivation, which was reversed by the addition of an excess of dithiothreitol. The NH2-terminal amino acid sequence of the enzyme contained the consensus sequence GXGXXG, indicative of the βαβ-fold of the flavin binding site and shared homologies with that of phenol 2-hydroxylase from Pseudomonas strain EST1001 as well as with that of 2,4-dichlorophenol 6-hydroxylase from Ralstonia eutropha.
No preview · Article · Oct 1997 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Sphingomonas herbicidovorans MH (previously designated Flavobacterium sp. strain MH) was able to utilize the chiral herbicide (RS)-2-(4-chloro-2-methylphenoxy)propionic acid (mecoprop) as the sole carbon and energy source. When strain MH was offered racemic mecoprop as the growth substrate, it could degrade both the (R) and the (S) enantiomer to completion, as shown by biomass formation, substrate consumption, and stoichiometric chloride release. However, the (S) enantiomer disappeared much faster from the culture medium than the (R) enantiomer. These results suggest the involvement of specific enzymes for the degradation of each enantiomer. This view was substantiated by the fact that resting cells of strain MH grown on (S)-mecoprop were able to degrade the (S) but not the (R) enantiomer of mecoprop. Accordingly, resting cells of strain MH grown on (R)-mecoprop preferentially metabolized the (R) enantiomer. Nevertheless, such cells could transform (S)-mecoprop at low rates. Oxygen uptake rates with resting cells confirmed the above view, as oxygen consumption was strongly dependent on the growth substrate. Cells grown on (R)-mecoprop showed oxygen uptake rates more than two times higher upon incubation with the (R) than upon incubation with the (S) enantiomer and vice versa.
Full-text · Article · Jan 1997 · Applied and Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: Rhodococcus rhodochrous PB1 was isolated from compost soil by selective culture with racemic 3-phenylbutyric acid as the sole carbon and energy source. Growth experiments with the single pure enantiomers as well as with the racemate showed that only one of the two enantiomers, (R)-3-phenylbutyric acid, supported growth of strain PB1. Nevertheless, (S)-3-phenylbutyric acid was cometabolically transformed to, presumably, (S)-3-(2,3-dihydroxyphenyl)butyric acid (the absolute configuration at the C-3 atom is not known yet) by (R)-3-phenylbutyric acid-grown cells of strain PB1, as shown by (sup1)H nuclear magnetic resonance spectroscopy of the partially purified compound and gas chromatography-mass spectrometry analysis of the trimethylsilyl derivative. Oxygen uptake rates suggest that either 3-phenylpropionic acid or cinnamic acid (trans-3-phenyl-2-propenoic acid) is the substrate for aromatic ring hydroxylation. This view is substantiated by the fact that 3-(2,3-dihydroxyphenyl)propionic acid was a substrate for meta cleavage in cell extracts of (R)-3-phenylbutyric acid-grown cells of strain PB1. Gas chromatography-mass spectrometry analysis of trimethylsilane-treated ethyl acetate extracts of incubation mixtures showed that both the meta-cleavage product, 2-hydroxy-6-oxo-2,4-nonadiene-1,9-dicarboxylic acid, and succinate, a hydrolysis product thereof, were formed during such incubations.
[Show abstract][Hide abstract] ABSTRACT: The chlorobenzene degradation pathway of Pseudomonas sp. strain P51 is an evolutionary novelty. The first enzymes of the pathway, the chlorobenzene dioxygenase and the cis-chlorobenzene dihydrodiol dehydrogenase, are encoded on a plasmid-located transposon Tn5280. Chlorobenzene dioxygenase is a four-protein complex, formed by the gene products of tcbAa for the large subunit of the terminal oxygenase, tcbAb for the small subunit, tcbAc for the ferredoxin, and tcbAd for the NADH reductase. Directly downstream of tcbAd is the gene for the cis-chlorobenzene dihydrodiol dehydrogenase, tcbB. Homology comparisons indicated that these genes and gene products are most closely related to those for toluene (todC1C2BAD) and benzene degradation (bedC1C2BA and bnzABCD) and distantly to those for biphenyl, naphthalene, and benzoate degradation. Similar to the tod-encoded enzymes, chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol dehydrogenase were capable of oxidizing 1,2-dichlorobenzene, toluene, naphthalene, and biphenyl, but not benzoate, to the corresponding dihydrodiol and dihydroxy intermediates. These data strongly suggest that the chlorobenzene dioxygenase and dehydrogenase originated from a toluene or benzene degradation pathway, probably by horizontal gene transfer. This evolutionary event left its traces as short gene fragments directly outside the tcbAB coding regions.
No preview · Article · Mar 1996 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Pseudomonas sp. strain HBP1 Prp grew on 2-isopropylphenol as the sole carbon and energy source with a maximal specific growth rate of 0.14 h-1 and transient accumulation of isobutyric acid. Oxygen uptake experiments with resting cells and enzyme assays with crude-cell extracts showed that 2-isopropylphenol was catabolized via a broad-spectrum meta cleavage pathway. These findings were confirmed by experiments with partially purified enzymes. Identification of 3-isopropylcatechol and 2-hydroxy-6-oxo-7-methylocta-2,4-dienoic acid as the products of the initial monooxygenase reaction and the subsequent extradiol ring cleavage dioxygenase reaction, respectively, was based on gas chromatography-mass spectrometry analysis of the corresponding trimethylsilyl derivatives. The meta cleavage product hydrolase hydrolyzed 2-hydroxy-6-oxo-7-methylocta-2,4-dienoic acid (meta cleavage product of 2-isopropylphenol) to isobutyric acid and 2-hydroxypent-2,4-dienoic acid.
Preview · Article · Jan 1995 · Applied and Environmental Microbiology