Dataset Information

Examination of the Glycine Betaine-Dependent Methylotrophic Methanogenesis Pathway: Insights Into Anaerobic Quaternary Amine Methylotrophy.

ABSTRACT: Recent studies indicate that environmentally abundant quaternary amines (QAs) are a primary source for methanogenesis, yet the catabolic enzymes are unknown. We hypothesized that the methanogenic archaeon Methanolobus vulcani B1d metabolizes glycine betaine (GB) through a corrinoid-dependent GB:coenzyme M (CoM) methyl transfer pathway. The draft genome sequence of M. vulcani B1d revealed a gene encoding a predicted non-pyrrolysine MttB homolog (MV8460) with high sequence similarity to the GB methyltransferase encoded by Desulfitobacterium hafniense Y51. MV8460 catalyzes GB-dependent methylation of free cob(I)alamin indicating it is an authentic MtgB enzyme. Proteomic analysis revealed that MV8460 and a corrinoid binding protein (MV8465) were highly abundant when M. vulcani B1d was grown on GB relative to growth on trimethylamine. The abundance of a corrinoid reductive activation enzyme (MV10335) and a methylcorrinoid:CoM methyltransferase (MV10360) were significantly higher in GB-grown B1d lysates compared to other homologs. The GB:CoM pathway was fully reconstituted in vitro using recombinant MV8460, MV8465, MV10335, and MV10360. Demonstration of the complete GB:CoM pathway expands the knowledge of direct QA-dependent methylotrophy and establishes a model to identify additional ecologically relevant anaerobic quaternary amine pathways.

SUBMITTER: Creighbaum AJ

PROVIDER: S-EPMC6855144 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Examination of the Glycine Betaine-Dependent Methylotrophic Methanogenesis Pathway: Insights Into Anaerobic Quaternary Amine Methylotrophy.

Creighbaum Adam J AJ Ticak Tomislav T Shinde Shrameeta S Wang Xin X Ferguson Donald J DJ

Frontiers in microbiology 20191107

Recent studies indicate that environmentally abundant quaternary amines (QAs) are a primary source for methanogenesis, yet the catabolic enzymes are unknown. We hypothesized that the methanogenic archaeon <i>Methanolobus vulcani</i> B1d metabolizes glycine betaine (GB) through a corrinoid-dependent GB:coenzyme M (CoM) methyl transfer pathway. The draft genome sequence of <i>M. vulcani</i> B1d revealed a gene encoding a predicted non-pyrrolysine MttB homolog (MV8460) with high sequence similarity ...[more]

PMID: 31787957

Similar Datasets

Project description:Microbial communities inhabiting the deep-sea salt-saturated anoxic lakes of the Eastern Mediterranean operate under harsh physical-chemical conditions that are incompatible with the lifestyle of common marine microorganisms. Here, we investigated a stable three-component microbial consortium obtained from the brine of the recently discovered deep-sea salt-saturated Lake Thetis. The trophic network of this consortium, established at salinities up to 240, relies on fermentative decomposition of common osmoprotectant glycine betaine (GB). Similarly to known extreme halophilic anaerobic GB-degrading enrichments, the initial step of GB degradation starts with its reductive cleavage to trimethylamine and acetate, carried out by the fermenting member of the Thetis enrichment, Halobacteroides lacunaris TB21. In contrast to acetate, which cannot be easily oxidized in salt-saturated anoxic environments, trimethylamine represents an advantageous C?-substrate for methylotrophic methanogenic member of the Thetis enrichment, Methanohalophilus sp. TA21. This second member of the consortium likely produces hydrogen via methylotrophic modification of reductive acetyl-CoA pathway because the initial anaerobic GB cleavage reaction requires the consumption of reducing equivalents. Ecophysiological role of the third member of the Thetis consortium, Halanaerobium sp. TB24, which lacks the capability of either GB or trimethylamine degradation, remains yet to be elucidated. As it is true for cultivated members of family Halanaerobiaceae, the isolate TB24 can obtain energy primarily by fermenting simple sugars and producing hydrogen as one of the end products. Hence, by consuming of TB21 and TA21 metabolites, Halanaerobium sp. TB24 can be an additional provider of reducing equivalents required for reductive degradation of GB. Description of the Thetis GB-degrading consortium indicated that anaerobic degradation of osmoregulatory molecules may play important role in the overall turnover of organic carbon in anoxic hypersaline biotopes.

Project description:Folate, or vitamin B9, is an important compound in one-carbon metabolism. Previous studies have found weaker binding of dihydrofolate to dihydrofolate reductase in the presence of osmolytes. In other words, osmolytes are more difficult to remove from the dihydrofolate solvation shell than water; this shifts the equilibrium toward the free ligand and protein species. This study uses vapor-pressure osmometry to explore the interaction of folate with the model osmolyte, glycine betaine. This method yields a preferential interaction potential (?23/RT value). This value is concentration-dependent as folate dimerizes. The ?23/RT value also tracks the deprotonation of folate's N3-O4 keto-enol group, yielding a pKa of 8.1. To determine which folate atoms interact most strongly with betaine, the interaction of heterocyclic aromatic compounds (as well as other small molecules) with betaine was monitored. Using an accessible surface area approach coupled with osmometry measurements, deconvolution of the ?23/RT values into ? values for atom types was achieved. This allows prediction of ?23/RT values for larger molecules such as folate. Molecular dynamics simulations of folate show a variety of structures from extended to L-shaped. These conformers possess ?23/RT values from -0.18 to 0.09 m-1, where a negative value indicates a preference for solvation by betaine and a positive value indicates a preference for water. This range of values is consistent with values observed in osmometry and solubility experiments. As the average predicted folate ?23/RT value is near zero, this indicates folate interacts almost equally well with betaine and water. Specifically, the glutamate tail prefers to interact with water, while the aromatic rings prefer betaine. In general, the more protonated species in our small molecule survey interact better with betaine as they provide a source of hydrogens (betaine is not a hydrogen bond donor). Upon deprotonation of the small molecule, the preference swings toward water interaction because of its hydrogen bond donating capacities.

Project description:Although some bacteria, including Chromohalobacter salexigens DSM 3043, can use glycine betaine (GB) as a sole source of carbon and energy, little information is available about the genes and their encoded proteins involved in the initial step of the GB degradation pathway. In the present study, the results of conserved domain analysis, construction of in-frame deletion mutants, and an in vivo functional complementation assay suggested that the open reading frames Csal_1004 and Csal_1005, designated bmoA and bmoB, respectively, may act as the terminal oxygenase and the ferredoxin reductase genes in a novel Rieske-type oxygenase system to convert GB to dimethylglycine in C. salexigens DSM 3043. To further verify their function, BmoA and BmoB were heterologously overexpressed in Escherichia coli, and 13C nuclear magnetic resonance analysis revealed that dimethylglycine was accumulated in E. coli BL21(DE3) expressing BmoAB or BmoA. In addition, His-tagged BmoA and BmoB were individually purified to electrophoretic homogeneity and estimated to be a homotrimer and a monomer, respectively. In vitro biochemical analysis indicated that BmoB is an NADH-dependent flavin reductase with one noncovalently bound flavin adenine dinucleotide (FAD) as its prosthetic group. In the presence of BmoB, NADH, and flavin, BmoA could aerobically degrade GB to dimethylglycine with the concomitant production of formaldehyde. BmoA exhibited strict substrate specificity for GB, and its demethylation activity was stimulated by Fe2+ Phylogenetic analysis showed that BmoA belongs to group V of the Rieske nonheme iron oxygenase (RO) family, and all the members in this group were able to use quaternary ammonium compounds as substrates.IMPORTANCE GB is widely distributed in nature. In addition to being accumulated intracellularly as a compatible solute to deal with osmotic stress, it can be utilized by many bacteria as a source of carbon and energy. However, very limited knowledge is presently available about the molecular and biochemical mechanisms for the initial step of the aerobic GB degradation pathway in bacteria. Here, we report the molecular and biochemical characterization of a novel two-component Rieske-type monooxygenase system, GB monooxygenase (BMO), which is responsible for oxidative demethylation of GB to dimethylglycine in C. salexigens DSM 3043. The results gained in this study extend our knowledge on the catalytic reaction of microbial GB degradation to dimethylglycine.

Dataset Information

Examination of the Glycine Betaine-Dependent Methylotrophic Methanogenesis Pathway: Insights Into Anaerobic Quaternary Amine Methylotrophy.

Publications

Examination of the Glycine Betaine-Dependent Methylotrophic Methanogenesis Pathway: Insights Into Anaerobic Quaternary Amine Methylotrophy.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets