The long term goal of this research is to characterize the metabolic regulation of pathways of folate metabolism in both normal and diseased states, and to determine reaction mechanisms for folate dependent-enzymes. We propose to continue our studies on methylenetetrahydrofolate reductase, a flavoprotein which regulates a key branch point in folate metabolism. This enzyme catalyzes the first step in the flow of one carbon units from methylenetetrahydrofolate into the pathway for de novo biosynthesis of methyl groups, and its activity is regulated by adenosylmethionine. Rapid reaction and steady-state kinetics, coupled with photoaffinity labelling of ligand binding sites, will be used to elucidate the mode of regulation of this enzyme. We propose to purify the next enzyme in the pathway, cobalamin-dependent methionine synthase, from pig liver and to study the activation of and catalysis by enzymes from both bacterial and mammalian sources. The inhibition of these enzymes by nitrous oxide (N20), a commonly used anaesthetic agent, will also be examined. The genes coding for both the cobalamin-dependent and cobalamin-independent methionine synthase enzymes from E. coli will be cloned and sequenced and evidence for the mechanism by which the cobalamin-dependent holoenzyme represses synthesis of the cobalamin-independent enzyme will be sought. Studies are also planned to investigate the effect of ZTP, a bacterial alarmone which signals folate deprivation, on the synthesis of two key enzymes in bacterial folate biosynthesis, IMP dehydrogenase and GTP cyclohydrolase.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024908-10
Application #
3272645
Study Section
Biochemistry Study Section (BIO)
Project Start
1978-04-01
Project End
1991-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
10
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Organized Research Units
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Koutmos, Markos; Gherasim, Carmen; Smith, Janet L et al. (2011) Structural basis of multifunctionality in a vitamin B12-processing enzyme. J Biol Chem 286:29780-7
Matthews, Rowena G (2009) Cobalamin- and corrinoid-dependent enzymes. Met Ions Life Sci 6:53-114
Liptak, Matthew D; Fleischhacker, Angela S; Matthews, Rowena G et al. (2009) Spectroscopic and computational characterization of the base-off forms of cob(II)alamin. J Phys Chem B 113:5245-54
Matthews, Rowena G; Koutmos, Markos; Datta, Supratim (2008) Cobalamin-dependent and cobamide-dependent methyltransferases. Curr Opin Struct Biol 18:658-66
Fleischhacker, Angela S; Matthews, Rowena G (2007) Ligand trans influence governs conformation in cobalamin-dependent methionine synthase. Biochemistry 46:12382-92
Huang, Sha; Romanchuk, Gail; Pattridge, Katherine et al. (2007) Reactivation of methionine synthase from Thermotoga maritima (TM0268) requires the downstream gene product TM0269. Protein Sci 16:1588-95
Pejchal, Robert; Campbell, Elizabeth; Guenther, Brian D et al. (2006) Structural perturbations in the Ala --> Val polymorphism of methylenetetrahydrofolate reductase: how binding of folates may protect against inactivation. Biochemistry 45:4808-18
Taurog, Rebecca E; Matthews, Rowena G (2006) Activation of methyltetrahydrofolate by cobalamin-independent methionine synthase. Biochemistry 45:5092-102
Yamada, Kazuhiro; Gravel, Roy A; Toraya, Tetsuo et al. (2006) Human methionine synthase reductase is a molecular chaperone for human methionine synthase. Proc Natl Acad Sci U S A 103:9476-81
Taurog, Rebecca E; Jakubowski, Hieronim; Matthews, Rowena G (2006) Synergistic, random sequential binding of substrates in cobalamin-independent methionine synthase. Biochemistry 45:5083-91

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