The PI proposes to continue and extend ongoing studies of the enzymology of microbial natural product biosynthesis. Among the metabolites to be examined are the macrolide antibiotics erythromycin (1) and methymycin (2), as well as pyridoxal phosphate (3, vitamin B6). A combination of chemical, enzymological, and molecular genetic techniques will be used to establish the molecular basis for the programming of the complex series of reactions responsible for polyketide chain elongation and to elucidate the final steps in the assembly of the pyridoxine ring of 3. It is expected that the results of these studies will be broadly applicable not only to the understanding of polyketide and other natural product biosynthetic processes in general, but will provide fundamental insights into how catalysis and molecular recognition control both product specificity and molecular diversity in Nature. 1) Deoxyerythronolide B Synthase (DEBS) is a multifunctional, modular protein which catalyzes the formation of 6-deoxyerythronolide B (4), the parent aglycone of erythromycin A. A truncated mutant, DEBS1+TE, a bimodular polyketide synthase that catalyzes the first two cycles of polyketide chain elongation leading to the formation of the triketide lactone 5, will be used as a key experimental model to examine the substrate specificity and synthetic versatility of the polyketide synthase, and to establish the role of the individual catalytic domains. 2) Recent results from our laboratory on the overexpression and characterization of EryK, a P450-dependent oxygenase that catalyzes the hydroxylation of C-12 erythromycin, will be extended to the study of the C-10 and C-12 hydroxylases that generate methymycin (s) and its cometabolite neomethymycin (6), respectively. 3) The PI will investigate the final steps in the formation of the characteristic pyridoxine ring of vitamin B6. Two proteins, PdxA and PdxJ, which have been previously implicated in the conversion of the 5-carbon sugar D-1-deoxyxylulose(7) and the amino acid anog 4-hydroxythreonine-4-phosphate (8-P) to pyridoxal phosphate (3), have been overexpressed and purified. The investigators will carry out incubations to confirm the proposed role of these two proteins and to determine the cofactor requirements and mechanism of the oxidative ring-forming reaction.
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