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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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8 NATURE CHEMICAL BIOLOGY | VOL 14 | JANUARY 2018 | www.nature.com/naturechemicalbiology
PUBLISHED ONLINE: 6 NOVEMBER 2017 | DOI: 10.1038/NCHEMBIO.2512
ARTICLE
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
β
ligand, a central cobalt-containing
corrin ring, and a lower Co
α
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
β
ligand
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: frank.loeffler@utk.edu or junyan@iae.ac.cn
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
a
-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.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
... 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. ...
Chapter
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. ...
Article
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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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Chapter
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Chapter
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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.
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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.
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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.
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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.