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Purinyl-cobamide is a Native Prosthetic Group of Reductive Dehalogenases

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Cobamides such as vitamin B12 are structurally conserved, cobalt-containing tetrapyrrole biomolecules that have essential biochemical functions in all domains of life. In organohalide respiration, a vital biological process for the global cycling of natural and anthropogenic organohalogens, cobamides are the requisite prosthetic groups for carbon–halogen bond-cleaving reductive dehalogenases. This study reports the biosynthesis of a new cobamide with unsubstituted purine as the lower base and assigns unsubstituted purine a biological function by demonstrating that Coα-purinyl-cobamide (purinyl-Cba) is the native prosthetic group in catalytically active tetrachloroethene reductive dehalogenases of Desulfitobacterium hafniense. Cobamides featuring different lower bases are not functionally equivalent, and purinyl-Cba elicits different physiological responses in corrinoid-auxotrophic, organohalide-respiring bacteria. Given that cobamide-dependent enzymes catalyze key steps in essential metabolic pathways, the discovery of a novel cobamide structure and the realization that lower bases can effectively modulate enzyme activities generate opportunities to manipulate functionalities of microbiomes.
| Phylogenetic analysis of CobT homologous proteins and substrate specificity of Dsf CobT. (a) the Dsf cobt activated dMb or purine to their respective α-ribazole-5′-phosphate (α-Rp) forms. naMn, nicotinic acid mononucleotide. (b) phylogenetic relationship of 40 cobt and homologous proteins from phylogenetically diverse corrinoid-auxotrophic and prototrophic bacteria. Solid black dots indicate cobt enzymes with biochemically determined substrate specificities. cobt enzymes of other members of the peptococcaceae may share catalytic features with the Dsf cobt and activate purine as a lower base for cobamide biosynthesis (indicated by the dashed line-enclosed pink area). (c) Hplc analysis demonstrating purified cobt activity with naMn and a lower base as the substrates. the small peak to the left of nicotinic acid in the bottom trace (Dhc cobt assay with purine) has a retention time similar to that of α-Rp [purine] shown in the panel above, but spectral analysis revealed distinct absorbance features. (d) the formation of α-Rp [dMb] or α-Rp [purine] in cobt assays with dMb or purine provided as a lower base substrate. the α-Rp concentrations were calculated on the basis of the concentration decreases of the respective lower base substrates. data are averages of measurements from duplicate assays. the (*) indicates that no α-Rp [purine] production was observed in Dhc cobt assays with purine as the lower base. (e) MS analysis of α-Rp [dMb] and α-Rp [purine] formed in cobt enzyme assays.
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Corrinoids are the most complicated organometallic cofactors
used in biology to catalyze essential biochemical reactions
including methyl group transfer, carbon skeleton rearrange-
ment and reductive dehalogenation1. Complete corrinoids (i.e., cob-
amides) consist of an upper Co
ligand, a central cobalt-containing
corrin ring, and a lower Co
base as part of the nucleotide loop that
is connected to the corrin ring2. For example, vitamin B12 (cyanoco-
balamin) carries the artificial cyano group as an upper Co
and 5,6-dimethylbenzimidazole (DMB) as the lower base (Fig. 1).
Variations in cobamide (Cba) structure exist in the upper ligand,
the lower base, and, in one documented case, the side chain in the
nucleotide loop3. Physiologically functional cobamides use a methyl
group or a 5-deoxyadenosyl moiety as the upper ligand and one of
16 known lower bases connected to the nucleotide loop2 (Fig. 1).
Reductive dehalogenation reactions can be mediated abiotically by
vitamin B12 and its analogs in the presence of a strong reductant
capable of generating the cobalt(I) supernucleophile4 or can be cata-
lyzed by corrinoid-dependent reductive dehalogenases (RDases)5.
In either case, the cobalt(I) supernucleophile is critical for the initia-
tion of carbon–halogen bond cleavage6.
Recently resolved RDase crystal structures of NpRdhA (PDB
ID 4RAS) from Nitratireductor pacificus and PceA (PDB ID 4UR0)
from Sulfurospirillum multivorans demonstrate that their prosthetic
groups coenzyme B12 and norpseudo-B12, respectively, are in the
base-off configurations, indicating that their respective lower bases,
DMB and adenine, are uncoordinated and distant from the redox-
active cobalt atom7,8. Therefore, the lower base is not assigned a
direct role in catalysis or electron transfer for restoring the reac-
tive cobalt(I) state. Contrary to expectations, lower-base structure
does impact reductive dechlorination rates and growth of obligate
organohalide-respiring, corrinoid-auxotrophic Dehalococcoides
mccartyi (Dhc) strains9,10. Maximum reductive dechlorination
activity and Dhc growth require the addition of cobamides carry-
ing DMB or 5-methylbenzimidazole (5-MeBza) as the lower base,
and cobamides with other lower-base structures compromise
growth (e.g., 5-methoxybenzimidazole, 5-OMeBza; benzimidazole,
Bza) or cause complete loss of reductive dechlorination activity
(e.g., 5-hydroxybenzimidazole, 5-OHBza; adenine)10,11. The lower
base structure also affects substrate utilization in the homoaceto-
gen Sporomusa ovata, which uses phenolic cobamide-dependent
methyltransferases for energy conservation and carbon assimila-
tion using the Wood–Ljungdahl pathway12. The addition of 5-Me-
Bza or DMB to the medium results in the formation of non-native
5-methylbenzimidazolyl-cobamide (5-MeBza-Cba) or cobalamin,
respectively, severely inhibiting growth with methanol or 3,4-dime-
thoxybenzoate as substrates12. These findings indicate that the lower
bases can affect enzyme activity and cause substantial consequences
to the host organisms; however, the mechanistic understanding is
lacking. To date, 16 naturally occurring lower bases have been iden-
tified, mostly from anaerobic bacteria, with the most recent struc-
ture, phenol in phenolyl-cobamide of Sporomusa ovata (Fig. 1),
discovered in 1989 (ref. 13).
Members of the genus Desulfitobacterium (Dsf) are strictly
anaerobic, metabolically versatile, Gram-positive bacteria common
in subsurface environments, where they contribute to the reductive
dehalogenation of organohalogens, including groundwater con-
taminants such as chlorinated solvents14,15. The tetrachloroethene
(PCE) RDases have a strict requirement for cobamide as a pros-
thetic group, and generate predominantly cis-1,2-dichloroethene
(cDCE) and inorganic chloride as products16. The characterized
1Department of Microbiology, University of Tennessee, Knoxville, Tennessee, USA. 2Key Laboratory of Pollution Ecology and Environmental Engineering,
Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, Liaoning, P.R. China. 3Center for Environmental Biotechnology, University of
Tennessee, Knoxville, Tennessee, USA. 4Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA. 5Joint Institute for Biological
Sciences (JIBS), Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA. 6Department of Chemistry, University of Tennessee, Knoxville, Tennessee,
USA. 7Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada. 8Bredesen Center for Interdisciplinary
Research and Graduate Education, University of Tennessee, Knoxville, Tennessee, USA. 9Department of Civil and Environmental Engineering, University
of Tennessee, Knoxville, Tennessee, USA. 10Department of Biosystems Engineering and Soil Science, University of Tennessee, Knoxville, Tennessee, USA.
*e-mail: or
Purinyl-cobamide is a native prosthetic group
of reductive dehalogenases
Jun Yan1–5*, Meng Bi1, Allen K Bourdon6, Abigail T Farmer6, Po-Hsiang Wang7, Olivia Molenda7,
Andrew T Quaile7, Nannan Jiang3–5,8, Yi Yang3,9, Yongchao Yin1, Burcu S¸ims¸ir3,9, Shawn R Campagna6 ,
Elizabeth A Edwards7 & Frank E Löffler1,3–5,8–10*
Cobamides such as vitamin B12 are structurally conserved, cobalt-containing tetrapyrrole biomolecules that have essential bio-
chemical functions in all domains of life. In organohalide respiration, a vital biological process for the global cycling of natural
and anthropogenic organohalogens, cobamides are the requisite prosthetic groups for carbon–halogen bond-cleaving reductive
dehalogenases. This study reports the biosynthesis of a new cobamide with unsubstituted purine as the lower base and assigns
unsubstituted purine a biological function by demonstrating that Co
-purinyl-cobamide (purinyl-Cba) is the native prosthetic
group in catalytically active tetrachloroethene reductive dehalogenases of Desulfitobacterium hafniense. Cobamides featuring
different lower bases are not functionally equivalent, and purinyl-Cba elicits different physiological responses in corrinoid-aux-
otrophic, organohalide-respiring bacteria. Given that cobamide-dependent enzymes catalyze key steps in essential metabolic
pathways, the discovery of a novel cobamide structure and the realization that lower bases can effectively modulate enzyme
activities generate opportunities to manipulate functionalities of microbiomes.
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
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... The numerous cobamide forms differ primarily in the structure of the lower ligand. For example, while 5,6-dimethylbenzimidazole (DMB) is the lower ligand of cobalamin, a variety of other benzimidazoles, purines, and phenolics have been found as lower ligands in cobamides isolated from natural environments or laboratory cultures ( Fig. 1C) (Renz, 1999;Yan et al., 2018). Often when a cobamide is bound by an enzyme, the lower ligand does not associate with the cobalt ion and so is not directly involved in catalysis (Bommer et al., 2014;Drennan, Huang, Drummond, Matthews, & Ludwig, 1994;Mancia et al., 1996), yet its structure can still influence the catalytic capability of the cobamide as a cofactor (Lengyel, Mazumder, & Ochoa, 1960;Poppe, Stupperich, Hull, Buckel, & Retey, 1997;Sokolovskaya et al., 2021). ...
... PseudoB 12 and [2-MeAde]Cba were the first two "alternate" cobamide cofactors identified, but in total, 15 other natural vitamin B 12 analogs have been described (Fig. 1) (Kr€ autler et al., 2003;Renz, 1999;Yan et al., 2018). These various cobamides are produced de novo by only a subset of bacteria and archaea. ...
... However, the majority of prokaryotic genomes have been found to encode at least one cobamide-dependent enzyme (Shelton et al., 2019) and thus, "sharing" of this nutrient is considered a critical functional interaction in microbial communities (Sokolovskaya, Shelton, & Taga, 2020). Importantly though, it appears that most microbes have specific preferences for only a few cobamide forms (Helliwell et al., 2016;Mok & Taga, 2013;Shelton, Lyu, & Taga, 2020;Yan et al., 2018;Yi et al., 2012). Therefore, deciphering how microbiomes are shaped by cobamides requires not only knowledge about which microbes produce and require them, but also an understanding of the relative cobamide preferences of each organism. ...
Cobamides are a family of enzyme cofactors that are required by organisms in all domains of life. Over a dozen cobamides exist in nature although only cobalamin (vitamin B12), the cobamide required by humans, has been studied extensively. Cobamides are exclusively produced by a subset of prokaryotes. Importantly, the bacteria and archaea that synthesize cobamides de novo typically produce a single type of cobamide, and furthermore, organisms that use cobamides are selective for certain cobamides. Therefore, a detailed understanding of the cobamide-dependent metabolism of an organism or microbial community of interest requires experiments performed with a variety of cobamides. A notable challenge is that cobalamin is the only cobamide that is commercially available at present. In this chapter, we describe methods to extract, purify, and quantify various cobamides from bacteria for use in laboratory experiments.
... Volumes of 40 and 200 mL (in 100 and 500-mL serum flasks, respectively) of medium were prepared where the head space was replaced by a mixture of N 2 /CO 2 (80/20%) as described earlier (Comensoli et al., 2017) with the modifications that cyanocobalamin was supplemented at 50 µM (final concentration) and that dicyanocobinamide was used instead of cyanocobalamin for the cultivation of D. restrictus strain PER-K23. Dicyanocobinamide was used here to allow D. restrictus to decorate the corrinoid cofactor with the lower ligand of its choice, likely with purine as suggested earlier (Wang et al., 2017), and in studies on Desulfitobacterium (Yan et al., 2018;Schubert et al., 2019). Completed medium was supplemented with acetic acid (5 mM) as carbon source, the head space replaced by a gas mixture of H 2 /CO 2 (80/20%) to provide H 2 as electron donor, and inoculated with 5% (v/v) of a preculture. ...
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Organohalide respiration (OHR) is a bacterial anaerobic process that uses halogenated compounds, e.g., tetrachloroethene (PCE), as terminal electron acceptors. Our model organisms are Dehalobacter restrictus strain PER-K23, an obligate OHR bacterium (OHRB), and Desulfitobacterium hafniense strain TCE1, a bacterium with a versatile metabolism. The key enzyme is the PCE reductive dehalogenase (PceA) that is encoded in the highly conserved gene cluster (pceABCT) in both above-mentioned strains, and in other Firmicutes OHRB. To date, the functions of PceA and PceT, a dedicated molecular chaperone for the maturation of PceA, are well defined. However, the role of PceB and PceC are still not elucidated. We present a multilevel study aiming at deciphering the stoichiometry of pceABCT individual gene products. The investigation was assessed at RNA level by reverse transcription and (quantitative) polymerase chain reaction, while at protein level, proteomic analyses based on parallel reaction monitoring were performed to quantify the Pce proteins in cell-free extracts as well as in soluble and membrane fractions of both strains using heavy-labeled reference peptides. At RNA level, our results confirmed the co-transcription of all pce genes, while the quantitative analysis revealed a relative stoichiometry of the gene transcripts of pceA, pceB, pceC, and pceT at ~ 1.0:3.0:0.1:0.1 in D. restrictus. This trend was not observed in D. hafniense strain TCE1, where no substantial difference was measured for the four genes. At proteomic level, an apparent 2:1 stoichiometry of PceA and PceB was obtained in the membrane fraction, and a low abundance of PceC in comparison to the other two proteins. In the soluble fraction, a 1:1 stoichiometry of PceA and PceT was identified. In summary, we show that the pce gene cluster is transcribed as an operon with, however, a level of transcription that differs for individual genes, an observation that could be explained by post-transcriptional events. Despite challenges in the quantification of integral membrane proteins such as PceB and PceC, the similar abundance of PceA and PceB invites to consider them as forming a membrane-bound PceA2B protein complex, which, in contrast to the proposed model, seems to be devoid of PceC.
Vitamin B12 (the cyanated form of cobalamin cofactors) is best known for its essential role in human health. In addition to its function in human metabolism, cobalamin also plays important roles in microbial metabolism and can impact microbial community function. Cobalamin is a member of the structurally diverse family of cofactors known as cobamides that are produced exclusively by certain prokaryotes. Cobamides are considered shared nutrients in microbial communities because the majority of bacteria that possess cobamide-dependent enzymes cannot synthesize cobamides de novo. Furthermore, different microbes have evolved metabolic specificity for particular cobamides, and therefore, the availability of cobamides in the environment is important for cobamide-dependent microbes. Determining the cobamides present in an environment of interest is essential for understanding microbial metabolic interactions. By examining the abundances of different cobamides in diverse environments, including 10 obtained in this study, we find that, contrary to its preeminence in human metabolism, cobalamin is relatively rare in many microbial habitats. Comparison of cobamide profiles of mammalian gastrointestinal samples and wood-feeding insects reveals that host-associated cobamide abundances vary and that fecal cobamide profiles differ from those of their host gastrointestinal tracts. Environmental cobamide profiles obtained from aquatic, soil, and contaminated groundwater samples reveal that the cobamide compositions of environmental samples are highly variable. As the only commercially available cobamide, cobalamin is routinely supplied during microbial culturing efforts. However, these findings suggest that cobamides specific to a given microbiome may yield greater insight into nutrient utilization and physiological processes that occur in these habitats.
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In bacteria, many essential metabolic processes are controlled by riboswitches, gene regulatory RNAs that directly bind and detect metabolites. Highly specific effector binding enables riboswitches to respond to a single biologically relevant metabolite. Cobalamin riboswitches are a potential exception because over a dozen chemically similar but functionally distinct cobalamin variants (corrinoid cofactors) exist in nature. Here, we measured cobalamin riboswitch activity in vivo using a Bacillus subtilis fluorescent reporter system and found, among 38 tested riboswitches, a subset responded to corrinoids promiscuously, while others were semiselective. Analyses of chimeric riboswitches and structural models indicate, unlike other riboswitch classes, cobalamin riboswitches indirectly differentiate among corrinoids by sensing differences in their structural conformation. This regulatory strategy aligns riboswitch-corrinoid specificity with cellular corrinoid requirements in a B. subtilis model. Thus, bacteria can employ broadly sensitive riboswitches to cope with the chemical diversity of essential metabolites. IMPORTANCE Some bacterial mRNAs contain a region called a riboswitch which controls gene expression by binding to a metabolite in the cell. Typically, riboswitches sense and respond to a limited range of cellular metabolites, often just one type. In this work, we found the cobalamin (vitamin B12) riboswitch class is an exception, capable of sensing and responding to multiple variants of B12-collectively called corrinoids. We found cobalamin riboswitches vary in corrinoid specificity with some riboswitches responding to each of the corrinoids we tested, while others responding only to a subset of corrinoids. Our results suggest the latter class of riboswitches sense intrinsic conformational differences among corrinoids in order to support the corrinoid-specific needs of the cell. These findings provide insight into how bacteria sense and respond to an exceptionally diverse, often essential set of enzyme cofactors.
Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile, TePN) is one of the most widely used fungicides all over the world. Its major environmental transformation product 4-hydroxy-chlorothalonil (4-hydroxy-2,5,6-trichloroisophthalonitrile, 4-OH-TPN) is more persistent, mobile, and toxic and is frequently detected at a higher concentration in various habitats compared to its parent compound TePN. Further microbial transformation of 4-OH-TPN has never been reported. In this study, we demonstrated that 4-OH-TPN underwent complete microbial reductive dehalogenation to 4-hydroxy-isophthalonitrile via 4-hydroxy-dichloroisophthalonitrile and 4-hydroxy-monochloroisophthalonitrile. 16S rRNA gene amplicon sequencing demonstrated that Dehalogenimonas species was enriched from 6% to 17-22% after reductive dechlorination of 77.24 μmol of 4-OH-TPN. Meanwhile, Dehalogenimonas copies increased by one order of magnitude and obtained a yield of 1.78 ± 1.47 × 108 cells per μmol Cl- released (N = 6), indicating that 4-OH-TPN served as the terminal electron acceptor for organohalide respiration of Dehalogenimonas species. A draft genome of Dehalogenimonas species was assembled through metagenomic sequencing, which harbors 30 putative reductive dehalogenase genes. Syntrophobacter, Acetobacterium, and Methanosarcina spp. were found to be the major non-dechlorinating populations in the microbial community, who might play important roles in the reductive dechlorination of 4-OH-TPN by the Dehalogenimonas species. This study first reports that Dehalogenimonas sp. can also respire on the seemingly dead-end product of TePN, paving the way to complete biotransformation of the widely present TePN and broadening the substrate spectrum of Dehalogenimonas sp. to polychlorinated hydroxy-benzonitrile.
Diclofenac (DCF) is a pharmaceutically active contaminant frequently found in aquatic ecosystems. The transformation pathways and microbiology involved in the biodegradation of DCF, particularly under anoxic conditions, remain poorly understood. Here, we demonstrated microbially mediated reductive dechlorination of DCF in anaerobic enrichment culture derived from contaminated river sediment. Over 90% of the initial 76.7 ± 3.6 μM DCF was dechlorinated at a maximum rate of 1.8 ± 0.3 μM day−1 during a 160 days’ incubation. Mass spectrometric analysis confirmed that 2-(2-((2-chlorophenyl)amino)phenyl)acetic acid (2-CPA) and 2- anilinophenylacetic acid (2-APA) were formed as the monochlorinated and nonchlorinated DCF transformation products, respectively. A survey of microbial composition and Sanger sequencing revealed the enrichment and dominance of a new Dehalogenimonas population, designated as Dehalogenimonas sp. strain DCF, in the DCFdechlorinating community. Following the stoichiometric conversion of DCF to 2-CPA (76.0 ± 2.1 μM) and 2-APA (3.7 ± 0.8 μM), strain DCF cell densities increased by 24.4 ± 4.4-fold with a growth yield of 9.0 ± 0.1 × 108 cells per μmol chloride released. Our findings expand the metabolic capability in the genus Dehalogenimonas and highlight the relevant roles of organohalide-respiring bacteria for the natural attenuation of halogenated contaminants of emerging concerns (e.g., DCF).
Temperature is a key factor affecting microbial activity and ecology. An increase in temperature generally increases rates of microbial processes up to a certain threshold, above which rates decline rapidly. In the subsurface, temperature of groundwater is usually stable and related to the annual average temperature at the surface. However, anthropogenic activities related to the use of the subsurface, e.g. for thermal heat management, foremost heat storage, will affect the temperature of groundwater locally. This mini-review intends to summarize the current knowledge on reductive dehalogenation activities of the chlorinated ethenes, common urban groundwater contaminants, at different temperatures. This includes an overview of activity and dehalogenation extent at different temperatures in laboratory isolates and enrichment cultures, the effect of shifts in temperature in micro- and mesocosm studies as well as observed biotransformation at different natural and induced temperatures at contaminated field sites. Furthermore, we address indirect effects on biotransformation, e.g. changes in fermentation, methanogenesis and sulfate reduction as competing or synergetic microbial processes. Finally, we address the current gaps in knowledge regarding bioremediation of chlorinated ethenes, microbial community shifts and bottlenecks for active combination with thermal energy storage, and necessities for bioaugmentation and/or natural re-populations after exposure to high temperature.
Enzymes catalyze a wide variety of reactions with exquisite precision under crowded conditions within cellular environments. When encountered with a choice of small molecules in their vicinity, even though most enzymes continue to be specific about the substrate they pick, some others are able to accept a range of substrates and subsequently produce a variety of products. The biosynthesis of Vitamin B12, an essential nutrient required by humans involves a multi-substrate α-phosphoribosyltransferase enzyme CobT that activates the lower ligand of B12. Vitamin B12 is a member of the cobamide family of cofactors which share a common tetrapyrrolic corrin scaffold with a centrally coordinated cobalt ion, and an upper and a lower ligand. The structural difference between B12 and other cobamides mainly arises from variations in the lower ligand, which is attached to the activated corrin ring by CobT and other downstream enzymes. In this chapter, we describe the steps involved in identifying and reconstituting the activity of new CobT homologs by deriving lessons from those previously characterized. We then highlight biochemical techniques to study the unique properties of these homologs. Finally, we describe a pairwise substrate competition assay to rank CobT substrate preference, a general method that can be applied for the study of other multi-substrate enzymes. Overall, the analysis with CobT provides insights into the range of cobamides that can be synthesized by an organism or a community, complementing efforts to predict cobamide diversity from complex metagenomic data.
Cobamides are a family of structurally-diverse cofactors which includes vitamin B12 and over a dozen natural analogs. Within the nucleotide loop structure, cobamide analogs have variable lower ligands that fall into three categories: benzimidazoles, purines, and phenols. The range of cobamide analogs that can be utilized by an organism is dependent on the specificity of its cobamide-dependent enzymes, and most bacteria are able to utilize multiple analogs but not all. Some bacteria have pathways for cobamide remodeling, a process in which imported cobamides are converted into compatible analogs. Here we discuss cobamide analog diversity and three pathways for cobamide remodeling, mediated by amidohydrolase CbiZ, phosphodiesterase CbiR, and some homologs of cobamide synthase CobS. Remodeling proteins exhibit varying degrees of specificity for cobamide substrates, reflecting different strategies to ensure that imported cobamides can be utilized.
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Cobamides are cobalt-containing cyclic tetrapyrroles involved in the metabolism of organisms from all domains of life but produced de novo only by some bacteria and archaea. The pathway is thought to involve up to 30 enzymes, five of which comprise the so-called “late” steps of cobamide biosynthesis. Two of these reactions activate the corrin ring, one activates the nucleobase, a fourth one condenses activated precursors, and a phosphatase yields the final product of the pathway. The penultimate step is catalyzed by a polytopic integral membrane protein, namely, the cobamide (5′-phosphate) synthase, also known as cobamide synthase. At present, the reason for the association of all putative and bona fide cobamide synthases to cell membranes is unclear and intriguing. Here, we show that, in Escherichia coli, elevated levels of cobamide synthase kill the cell by dissipating the proton motive force and compromising membrane stability. We also show that overproduction of the phosphatase that catalyzes the last step of the pathway or phage shock protein A prevents cell death when the gene encoding cobamide synthase is overexpressed. We propose that in E. coli, and probably all cobamide producers, cobamide synthase anchors a multienzyme complex responsible for the assembly of vitamin B12 and other cobamides. IMPORTANCE E. coli is the best-studied prokaryote, and some strains of this bacterium are human pathogens. We show that when the level of the enzyme that catalyzes the penultimate step of vitamin B12 biosynthesis is elevated, the viability of E. coli decreases. These findings are of broad significance because the enzyme alluded to is an integral membrane protein in all cobamide-producing bacteria, many of which are human pathogens. Our results may provide new avenues for the development of antimicrobials, because none of the enzymes involved in vitamin B12 biosynthesis are present in mammalian cells.
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Two novel chlorinated alkane-respiring Dehalobacter restrictus strains CF and DCA were isolated from the same enrichment culture, ACT-3, and characterized. The closed genomes of these highly similar sister strains were previously assembled from metagenomic sequence data and annotated. The isolation of the strains enabled experimental verification of predicted annotations, particularly focusing on irregularities or predicted gaps in central metabolic pathways and cofactor biosynthesis. Similar to D. restrictus strain PER-K23, strains CF and DCA require arginine, histidine and threonine for growth, although the corresponding biosynthesis pathways are predicted to be functional. Using strain CF to experimentally verify annotations, we determined that the predicted defective serine biosynthesis pathway can be rescued with a promiscuous serine hydroxymethyltransferase. Strain CF grew without added thiamine although the thiamine biosynthesis pathway is predicted to be absent; intracellular thiamine diphosphate, the cofactor of carboxylases in central metabolism, was not detected in cell extracts. Thus, strain CF may use amino acids to replenish central metabolites, portending entangled metabolite exchanges in ACT-3. Consistent with annotation, strain CF possesses a functional corrinoid biosynthesis pathway, demonstrated by increasing corrinoid content during growth and guided cobalamin biosynthesis in corrinoid-free medium. Chloroform toxicity to corrinoid-producing methanogens and acetogens may drive the conservation of corrinoid autotrophy in Dehalobacter strains. Heme detection in strain CF cell extracts suggests the ‘archaeal’ heme biosynthesis pathway also functions in anaerobic Firmicutes. This study reinforces the importance of incorporating enzyme promiscuity and cofactor availability in genome-scale functional predictions and identifies essential nutrient interdependencies in anaerobic dechlorinating microbial communities.
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DUF89 family proteins occur widely in both prokaryotes and eukaryotes, but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), with subfamily II being split into stand-alone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metal-dependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027W revealed a novel phosphatase active site with fructose 6-phosphate and Mg(2+) bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.
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Virtually, all tumor cells as well as all immune cells express plasma membrane receptors for extracellular nucleosides (adenosine) and nucleotides (ATP, ADP, UTP, UDP and sugar UDP). The tumor microenvironment is characterized by an unusually high concentration of ATP and adenosine. Adenosine is a major determinant of the immunosuppressive tumor milieu. Sequential hydrolysis of extracellular ATP catalyzed by CD39 and CD73 is the main pathway for the generation of adenosine in the tumor interstitium. Extracellular ATP and adenosine mold both host and tumor responses. Depending on the specific receptor activated, extracellular purines mediate immunosuppression or immunostimulation on the host side, and growth stimulation or cytotoxicity on the tumor side. Recent progress in this field is providing the key to decode this complex scenario and to lay the basis to harness the potential benefits for therapy. Preclinical data show that targeting the adenosine-generating pathway (that is, CD73) or adenosinergic receptors (that is, A2A) relieves immunosuppresion and potently inhibits tumor growth. On the other hand, growth of experimental tumors is strongly inhibited by targeting the P2X7 ATP-selective receptor of cancer and immune cells. This review summarizes the recent data on the role played by extracellular purines (purinergic signaling) in host–tumor interaction and highlights novel therapeutic options stemming from recent advances in this field.
Dehalococcoides mccartyi strain CBDB1 is an obligate organohalide-respiring bacterium using only hydrogen as electron donor and halogenated organics as electron acceptor. Here, we studied proteins involved in the respiratory chain under non-denaturing conditions. Using Blue Native gel electrophoresis (BN-PAGE), gel filtration and ultrafiltration an active dehalogenating protein complex with a molecular mass of 250-270 kDa was identified. The active subunit of reductive dehalogenase (RdhA) co-localized with a complex iron-sulfur molybdoenzyme (CISM) subunit (CbdbA195) and an iron-sulfur cluster containing subunit (CbdbA131) of the hydrogen uptake hydrogenase (Hup). No co-localization between the catalytically active subunits of hydrogenase and reductive dehalogenase was found. By two-dimensional BN/SDS-PAGE the stability of the complex towards detergents was assessed, demonstrating stepwise disintegration with increasing detergent concentrations. Chemical cross-linking confirmed the presence of a higher molecular mass reductive dehalogenase protein complex composed of RdhA, CISM I and Hup hydrogenase and proved to be a potential tool for stabilizing protein-protein interactions of the dehalogenating complex prior to membrane solubilization. Taken together, the identification of the respiratory dehalogenase protein complex and the absence of indications for quinone participation in the respiration suggest a quinone-independent protein-based respiratory electron transfer chain in D. mccartyi.
The quantum chemical cluster approach was used to elucidate the reaction mechanism of debromination catalyzed by the B12-dependent reductive dehalogenase NpRdhA. Various pathways, involving different oxidation states of the cobalt ion and different protonation states of the model, have been analyzed in order to find the most favorable one. We find that the reductive C-Br cleavage takes place exclusively at the CoI state via a heterolytic pathway in the singlet state. Importantly, the C-H bond formation and the C-Br bond cleavage proceeds via a concerted transition state, as opposed to the stepwise pathway suggested before. C-Br cleavage at the CoII state has a very high barrier, and the reduction of CoI to Co0 is associated with a very negative potential; thus, reductive dehalogenation at CoII and Co0 can be safely ruled out. Examination of substrates with different halogen substitutions (F, Cl, Br, I) shows that the dehalogenation reactivity follows the order C-I > C-Br > C-Cl > C-F, and the barrier for defluorination is so high that NpRdhA cannot catalyze that reaction.
Corrinoid auxotrophic organohalide-respiring Dehalococcoides mccartyi (Dhc) strains are keystone bacteria for reductive dechlorination of toxic and carcinogenic chloroorganic contaminants. We demonstrate that the lower base attached to the essential corrinoid cofactor of reductive dehalogenase (RDase) enzyme systems modulates dechlorination activity and affects the vinyl chloride (VC) RDases BvcA and VcrA differently. Amendment of 5,6-dimethylbenzimidazolyl-cobamide (DMB-Cba) to Dhc strain BAV1 and strain GT cultures supported cis-1,2-dichloroethene-to-ethene reductive dechlorination at rates of 107.0 (±12.0) μM and 67.4 (±1.4) μM Cl(-) released per day, respectively. Strain BAV1, expressing the BvcA RDase, reductively dechlorinated VC to ethene, although at up to fivefold lower rates in cultures amended with cobamides carrying 5-methylbenzimidazole (5-MeBza), 5-methoxybenzimidazole (5-OMeBza) or benzimidazole (Bza) as the lower base. In contrast, strain GT harboring the VcrA RDase failed to grow and dechlorinate VC to ethene in medium amended with 5-OMeBza-Cba or Bza-Cba. The amendment with DMB to inactive strain GT cultures restored the VC-to-ethene-dechlorinating phenotype and intracellular DMB-Cba was produced, demonstrating cobamide uptake and remodeling. The distinct responses of Dhc strains with BvcA versus VcrA RDases to different cobamides implicate that the lower base exerts control over Dhc reductive dechlorination rates and extents (that is, detoxification), and therefore the dynamics of Dhc strains with discrete reductive dechlorination capabilities. These findings emphasize that the role of the corrinoid/lower base synthesizing community must be understood to predict strain-specific Dhc activity and achieve efficacious contaminated site cleanup.The ISME Journal advance online publication, 10 November 2015; doi:10.1038/ismej.2015.197.
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.