CRYSTAL STRUCTURE OF SERINE HYDROXYMETHYLTRANSFERASE

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Serine hydroxymethyl transferase (SHMT) is a ubiquitous enzyme which lies at the center of one carbon metabolism in both prokaryotes and eukaryotes. One carbon units carried in any of three oxidation states by SHMT are contribute to the biosynthesis of purines, thymidylate, phospholipids, and methionine. SHMT is a pyridoxal phosphate enzyme and also requires tetrahydrofolate as a cofactor, the latter of which carries the one carbon units. The differential absorbance spectra of various intermediates has made it possible to characterize some of the steps of the reaction pathway. Beside the generation of one carbon units from the transformation of serine into glycine, SHMT has also been shown to catalyse a large number of other, mechanistically related reactions with different substages. The lack of a crystal structure of SHMT has hampered a fuller investigation of its mechanism and roles of functional groups in the enzyme. The aims of the work proposed here are to determine the crystal structure of E. coli SHMT by x-ray diffraction methods and to use this structure, its complexes with coenzymes, substrates and inhibitors, and related mutant structures to understand the mechanism of this enzyme. Crystals of plasmid-expressed E. coli SHMT complexed with 5-formyltetrahydrofolate and glycine have been obtained, which diffract to a resolution 2.8A and these will be used to solve the structure by the heavy atom isomorphous replacement method. Site mutants of this SHMT have already been created, and others can be made by mutagenesis for the study of the role of specific residues in the catalytic reaction. In the cycle for thymidylate biosynthesis, SHMT is the only one of the three enzymes of the cycle whose structure has not been determined. The other two are dihydrofolate reductase and thymidylate synthase, both of which have been targets for the design of chemotherapeutic anticancer compounds. Relatively little is known about the structures of the enzymes in the other two metabolic cycles in which SHMT is active. The centrality of SHMT to the critical pathway of one carbon metabolism makes it a fruitful prospect for the design of other compounds of potential therapeutic value. Realization of this potential will required a knowledge of its structure and an understanding of its mechanism, which are the objects of the work proposed here.