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, protein-protein interactions, multienzyme complex systems, and to investigate the integration of this pathway into the metabolic network. Comprehensive knowledege of the pathway and its components hat can ultimately be used in antimicrobial design and anti-cancer drug development. We will expand the multifaceted approach we have used for years to include metabolomics approaches to investigate the several aspects of coenzyme B12 biosynthesis that remain unresolved. We propose to do four things: i) to perform in-depth studies of the mechanism of function of the recently discovered, Fe-S-containing, oxygen-labile EutT corrinoid adenosyltransferase, and to understand the role of the metal in EutT catalysis; ii) to Identify genes of Salmonella that encode enzymes that synthesize 5,6-dimethylbenzimidazole (DMB, the lower ligand base of AdoCbl), and alpha-ribazole, the DMB nucleoside in the absence of the two known base-activating enzymes. We have strong genetic evidence of the existence of these unprecedented functions; iii) to identify the genes encoding the enzymes of the anaerobic of DMB biosynthetic pathway found in many human pathogens, and study the function of their products in vitro and in vivo. The analysis of this pathway has remained unexplored; and iv) to analyze the metabolome of strains of Salmonella that have been engineered to accumulate B12 intermediates that have only been studied in vitro. We will continue to collaborate with structural biologists led by Ivan Rayment (UW-Madison), with transition-metal spectroscopists led by Thomas Brunold (UW-Madison), and with mass spectrometrists led by Shawn Campagna (U of Tennessee-Knoxville). We expect to discover new enzymes and pathways, to understand their mechanism of catalysis, with the ultimate goal of understanding the function of the predicted membrane-associated multienzyme complex in vivo and vitro.

Public Health Relevance

Only prokaryotes make CoB12, and many of which are human pathogens. Precise knowledge of the biochemistry underpinning the pathway and an understanding of the structural properties of the enzymes involved, is critical to the design of antimicrobials that would have no side effects on the host and could be used to target disease-causing bacteria. The use of B12 bioconjugates to fight cancer is also very promising.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM040313-27
Application #
9116856
Study Section
Special Emphasis Panel (NSS)
Program Officer
Barski, Oleg
Project Start
1988-09-01
Project End
2020-07-31
Budget Start
2016-08-01
Budget End
2017-07-31
Support Year
27
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Georgia
Department
Type
DUNS #
004315578
City
Athens
State
GA
Country
United States
Zip Code
30602
Tavares, Norbert K; VanDrisse, Chelsey M; Escalante-Semerena, Jorge C (2018) Rhodobacterales use a unique L-threonine kinase for the assembly of the nucleotide loop of coenzyme B12. Mol Microbiol 110:239-261
Costa, Flavia G; Escalante-Semerena, Jorge C (2018) A New Class of EutT ATP:Co(I)rrinoid Adenosyltransferases Found in Listeria monocytogenes and Other Firmicutes Does Not Require a Metal Ion for Activity. Biochemistry 57:5076-5087
Mattes, Theodoric A; Escalante-Semerena, Jorge C (2018) Facile isolation of ?-ribazole from vitamin B12 hydrolysates using boronate affinity chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 1090:52-55
Stracey, Nuru G; Costa, Flavia G; Escalante-Semerena, Jorge C et al. (2018) Spectroscopic Study of the EutT Adenosyltransferase from Listeria monocytogenes: Evidence for the Formation of a Four-Coordinate Cob(II)alamin Intermediate. Biochemistry 57:5088-5095
Tavares, Norbert K; Zayas, Carmen L; Escalante-Semerena, Jorge C (2018) The Methanosarcina mazei MM2060 Gene Encodes a Bifunctional Kinase/Decarboxylase Enzyme Involved in Cobamide Biosynthesis. Biochemistry 57:4478-4495
Pallares, Ivan G; Moore, Theodore C; Escalante-Semerena, Jorge C et al. (2017) Spectroscopic Studies of the EutT Adenosyltransferase from Salmonella enterica: Evidence of a Tetrahedrally Coordinated Divalent Transition Metal Cofactor with Cysteine Ligation. Biochemistry 56:364-375
Mattes, Theodoric A; Escalante-Semerena, Jorge C (2017) Salmonella enterica synthesizes 5,6-dimethylbenzimidazolyl-(DMB)-?-riboside. Why some Firmicutes do not require the canonical DMB activation system to synthesize adenosylcobalamin. Mol Microbiol 103:269-281
Tavares, Norbert K; Escalante-Semerena, Jorge C (2017) A snapshot of evolution in action: emergence of new heme transport function derived from a coenzyme B12 biosynthetic enzyme. Environ Microbiol 19:8-10
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
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

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