The long-term goal is to relate molecular structure to specificity and reactivity in selected enzymes, with an emphasis on enzymes that require vitamin-based cofactors or essential metals. Three of the enzymes to be studied, B12-dependent methionine synthase (MetH), methylenetetrahydrofolate reductase (MTHFR), and betaine-homocysteine methyltransferase (BHMT), are key catalysts in the metabolism of one-carbon units and control the cellular and plasma levels of homocysteine and methionine. These metabolites are believed to be important indicators of risk for cardiovascular disease and neural tube defects. High-resolution x-ray analysis will be the primary tool to examine local and global conformation changes that are fundamental features of catalysis and control in these enzymes. Mutagenesis and biophysical techniques will also be employed to determine how these enzymes exploit conformation changes and how mutation leads to dysfunction. ? ? Descriptions of domain rearrangements will be obtained for B12-dependent methionine synthase (MetH), a prototype for complex proteins that undergo large domain movements. In MetH from E. coli, a large enzyme with four ligand binding modules, the B12-binding domain is required to move long distances to interact in turn with homocysteine, methyltetrahydrofolate, and S-adenosylmethionine. Structures of each module were determined earlier; the current goal is to understand how the modules interact and what drives their movements. Impairment of human methionine synthase, an ortholog of the E. coli enzyme, is responsible for many of the manifestations of B12 deficiency. Comparisons will be made with B12-independent methionine synthase, which catalyzes methylation of homocysteine without requiring an intermediate methyl carrier. ? ? Structure-function studies of human BHMT, an important enzyme in homocysteine homeostasis in liver, are aimed at understanding the conformation changes that produce ordered binding of substrates. ? ? The structure of methylenetetrahydrofolate reductase (MTHFR) from E. coli has been determined as a model for the catalytic module of human MTHFR, an enzyme that lies at a critical branch point in one-carbon metabolism and is allosterically controlled by S-adenosylmethionine. The bacterial model has been used to show how folates protect the activity of MTHFR, thereby lowering homocysteine levels. The larger human enzyme, containing both catalytic and regulatory modules, has now been expressed by our collaborators; structural studies are aimed at understanding the regulatory mechanisms. ? ?
