S-adenosylmethionine (AdoMet) plays essential roles in the metabolism of all cellular organisms, with functions that are commonly altered in diseases. The enzymes of AdoMet metabolism are targets for development of chemotherapeutic agents. The goals of this research are to elucidate the functioning of S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase, MAT), and of AdoMet decarboxylase (AdoMetDC) whose reaction commits AdoMet to polyamine synthesis. A plethora of experimental and computational techniques will provide new structural and mechanistic information regarding enzyme function, and lead towards discovery of novel inhibitors. The mechanism of MAT will be characterized by computational methods to elucidate the basis of catalysis of this two-step reaction. Experimental data guide the use of quantum mechanics in investigation of the roles of active site residues. Mechanistic deductions will be tested experimentally. Novel MAT inhibitors will be discovered by virtual docking of libraries of small molecules to the MAT crystal structure. Candidate inhibitors will be experimentally evaluated to identify compounds able to modulate cellular AdoMet levels. The human pathogen Streptococcus pyogenes has both a type of MAT typically found only in archaea and the bacterial form. The archaeal type MAT is hypothesized to synthesize novel metabolites using substrates in addition to ATP; this could provide a new antibiotic target. Both cloned S. pyogenes MATs will be expressed in E. coli, purified and characterized. If the specificity hypothesis is confirmed in vitro, extracts of S. pyogenes will reveal if these novel metabolites form in vivo. The apparently diverse catalytic mechanisms of two non-homologous pyruvoyl cofactor containing AdoMetDC will be contrasted. Kinetic and structural studies will elucidate the mechanisms of a representative of the activator independent class and a representative of the metal ion dependent group. The influence of the protein (and metal ion) on the electronic environment of the cofactor and the enzyme substrate complexes will be revealed from 13C and 15N NMR of complexes of selectively enriched protein and substrates. These studies will unmask why nature conserves use of a pyruvoyl cofactor for this metabolic function instead of adopting the common pyridoxal cofactor.
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