A central question in biology is how a cell efficiently integrates the large number of pathways that comprise intermediary metabolism. Although it is clear that some metabolites are generated in a regulated manner to maintain physiological balance, it is less clear how the cell achieves efficient synthesis of molecules whose steady intracellular level is very low, such as coenzymes and their precursors. The concept of channeling metabolites through multienzymatic complexes (a.k.a. metabolons) is particularly attractive to the analysis of major biosynthetic pathways such as the one synthesizing adenosyl-cobalamin (Ado-CBL), because this important coenzyme is made in very small amounts. We are using the late steps of Ado-CBL biosynthesis as a model system for probing the metabolon hypothesis. We have previously reported genetic evidence suggesting that enzymes catalyzing these steps may interact, and that these interactions may indeed be needed for metabolite channeling. Towards that goal we have established the biochemical role of 3 of the 4 enzymes thought to comprise the metabolon, and initiated collaborative work with Dr. Ivan Rayment (Enzyme Institute, UW-Madison) to pursue the crystallographic analysis of these proteins. Work represented in this proposal will continue the biochemical characterization of these enzymes. Our multidisciplinary approach to understanding the cobalamin biosynthetic pathway will make important contributions to our knowledge of broad, basic biochemical concepts such as protein/protein interactions, protein/coenzyme interactions, enzymology, multienzyme complex formation and metabolite channeling. This basic knowledge will help us explain in molecular terms the complex phenotypes of mutants deficient in cobalamin synthesis and the impact of the cobalamin biosynthetic pathway on cell physiology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM040313-10
Application #
2838547
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1988-09-01
Project End
2001-11-30
Budget Start
1998-12-01
Budget End
1999-11-30
Support Year
10
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Microbiology/Immun/Virology
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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
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
Newmister, Sean A; Otte, Michele M; Escalante-Semerena, Jorge C et al. (2011) Structure and mutational analysis of the archaeal GTP:AdoCbi-P guanylyltransferase (CobY) from Methanocaldococcus jannaschii: insights into GTP binding and dimerization. Biochemistry 50:5301-13
Mera, Paola E; Escalante-Semerena, Jorge C (2010) Dihydroflavin-driven adenosylation of 4-coordinate Co(II) corrinoids: are cobalamin reductases enzymes or electron transfer proteins? J Biol Chem 285:2911-7
Gray, Michael J; Escalante-Semerena, Jorge C (2010) A new pathway for the synthesis of ?-ribazole-phosphate in Listeria innocua. Mol Microbiol 77:1429-38
Claas, Kathy R; Parrish, J R; Maggio-Hall, L A et al. (2010) Functional analysis of the nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) enzyme, involved in the late steps of coenzyme B12 biosynthesis in Salmonella enterica. J Bacteriol 192:145-54
Otte, Michele M; Escalante-Semerena, Jorge C (2009) Biochemical characterization of the GTP:adenosylcobinamide-phosphate guanylyltransferase (CobY) enzyme of the hyperthermophilic archaeon Methanocaldococcus jannaschii. Biochemistry 48:5882-9
Gray, Michael J; Escalante-Semerena, Jorge C (2009) The cobinamide amidohydrolase (cobyric acid-forming) CbiZ enzyme: a critical activity of the cobamide remodelling system of Rhodobacter sphaeroides. Mol Microbiol 74:1198-210
Gray, Michael J; Escalante-Semerena, Jorge C (2009) In vivo analysis of cobinamide salvaging in Rhodobacter sphaeroides strain 2.4.1. J Bacteriol 191:3842-51
Mera, Paola E; St Maurice, Martin; Rayment, Ivan et al. (2009) Residue Phe112 of the human-type corrinoid adenosyltransferase (PduO) enzyme of Lactobacillus reuteri is critical to the formation of the four-coordinate Co(II) corrinoid substrate and to the activity of the enzyme. Biochemistry 48:3138-45

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