Coenzyme B12 (aka, adenosylcobalamin, AdoCbl) biosynthesis is a major biosynthetic pathway (>25 genes) that is unique to prokaryotes. Given the complex chemistry required to assemble this coenzyme, studies of its biosynthesis offer an opportunity to learn complex enzymology and to investigate how this large pathway is integrated with other metabolic processes in the cell. Coenzyme B12 is exclusively synthesized by prokaryotes, and it is essential to the survival of many pathogens. Hence, precise knowledge of the biochemistry underpinning this major pathway, and an understanding of the structural properties of the enzymes involved, is critical to the design of inhibitors that could be used to target disease-causing bacteria or any other prokaryote of societal importance. We will apply a multifaceted approach to continue our studies of AdoCbl biosynthesis. We propose to do three things: i) an in-depth studies of the recently discovered, Fe-S-containing, oxygen-labile EutT corrinoid adenosyltransferase;ii) we will dissect the function of the CbiZ enzyme, whose role in lower ligand remodeling was recently uncovered by our group;iii) we will characterize one function of a recently-discovered 5,6-dimethylbenzimidazole-riboside salvaging system found in important human pathogens. This part of the proposed work includes the initial analysis of identify the genes that encode the enzymes of the anaerobic of the lower ligand base, which in cobalamin is 5,6-dimethylbenzimidazole, a pathway that remains unexplored. We will continue to collaborate with structural biologists led by Ivan Rayment (Department of Biochemistry, UW-Madison), and with transition-metal spectroscopists led by Thomas Brunold (Chemistry Department, UW-Madison). Together, we will gain molecular insights into the assembly of this complex coenzyme and its physiological roles in all cells.

Public Health Relevance

Coenzyme B12 is exclusively synthesized by prokaryotes, and it is essential to the survival of animals, including humans. Precise knowledge of the biochemistry underpinning the biosynthesis of coenzyme B12, and an understanding of the structural properties of the enzymes involved, is critical to the design of inhibitors that could be used to target disease-causing bacteria or any other prokaryote of societal importance. Recent collaborative efforts between the PI and Swiss researchers highlight the impact of knowledge gained from the studies of coenzyme B12 synthesis in our fight against cancer. A multifaceted approach will be applied to learn more about the conversion of the vitamin to its coenzyme form, to elucidate biosynthetic strategies for the lower ligand base in strict anaerobes, and to reveal how prokaryotes remodel the end product of the pathway to suit their metabolic needs.

Agency
National Institute of Health (NIH)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM040313-25
Application #
8703712
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Barski, Oleg
Project Start
Project End
Budget Start
Budget End
Support Year
25
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Georgia
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
City
Athens
State
GA
Country
United States
Zip Code
30602
Pallares, Ivan G; Moore, Theodore C; Escalante-Semerena, Jorge C et al. (2016) Spectroscopic Studies of the EutT Adenosyltransferase from Salmonella enterica: Mechanism of Four-Coordinate Co(II)Cbl Formation. J Am Chem Soc 138:3694-704
Mattes, Theodoric A; Escalante-Semerena, Jorge C (2016) Salmonella enterica synthesizes 5,6-dimethylbenzimidazolyl-(DMB)-α-riboside. Why some Firmicutes do not require the canonical DMB activation system to synthesize adenosylcobalamin. Mol Microbiol :
Park, Kiyoung; Mera, Paola E; Escalante-Semerena, Jorge C et al. (2016) Resonance Raman spectroscopic study of the interaction between Co(II)rrinoids and the ATP:corrinoid adenosyltransferase PduO from Lactobacillus reuteri. J Biol Inorg Chem 21:669-81
Keller, Sebastian; Treder, Aaron; von Reuss, Stephan H et al. (2016) The SMUL_1544 Gene Product Governs Norcobamide Biosynthesis in the Tetrachloroethene-Respiring Bacterium Sulfurospirillum multivorans. J Bacteriol 198:2236-43
Moore, Theodore C; Escalante-Semerena, Jorge C (2016) The EutQ and EutP proteins are novel acetate kinases involved in ethanolamine catabolism: physiological implications for the function of the ethanolamine metabolosome in Salmonella enterica. Mol Microbiol 99:497-511
Park, Kiyoung; Mera, Paola E; Moore, Theodore C et al. (2015) Unprecedented Mechanism Employed by the Salmonella enterica EutT ATP:Co(I)rrinoid Adenosyltransferase Precludes Adenosylation of Incomplete Co(II)rrinoids. Angew Chem Int Ed Engl 54:7158-61
Singarapu, Kiran K; Otte, Michele M; Tonelli, Marco et al. (2015) Solution Structural Studies of GTP:Adenosylcobinamide-Phosphateguanylyl Transferase (CobY) from Methanocaldococcus jannaschii. PLoS One 10:e0141297
Chan, Chi Ho; Newmister, Sean A; Talyor, Keenan et al. (2014) Dissecting cobamide diversity through structural and functional analyses of the base-activating CobT enzyme of Salmonella enterica. Biochim Biophys Acta 1840:464-75
Pallares, Ivan G; Moore, Theodore C; Escalante-Semerena, Jorge C et al. (2014) Spectroscopic studies of the Salmonella enterica adenosyltransferase enzyme SeCobA: molecular-level insight into the mechanism of substrate Cob(II)alamin activation. Biochemistry 53:7969-82
Moore, Theodore C; Mera, Paola E; Escalante-Semerena, Jorge C (2014) the Eutt enzyme of Salmonella enterica is a unique ATP:Cob(I)alamin adenosyltransferase metalloprotein that requires ferrous ions for maximal activity. J Bacteriol 196:903-10

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