Structural Biology of Complex Enzymes

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Project Summary Natural product biosynthetic pathways harbor a vast synthetic potential for chemical groups that are rare in biology. Enzymes that were adapted from primary metabolism to novel functions in these secondary- metabolite pathways are a major source biocatalytic potential for many applications. In this project, the structure and function of several enzymes that catalyze unusual reactions in the biosynthesis of natural products will be investigated. Protein crystal structures will inform the development of functional assays, which will be used to test structure-based hypotheses about mechanism, substrate selectivity and product specificity. The enzymes are from bacterial and fungal biosynthetic pathways for several natural product classes, including polyketides, nonribosomal peptides and azaphilones. The four enzyme systems have great potential for biocatalyst development within or outside their natural pathways, for advancing our understanding of protein evolution in nature, for expanding the scope of enzyme chemistry, and for engineering new function. In the first system, a bacterial enzyme has distinct acyl transfer and decarboxylation functions in two biosynthetic pathways. The dual functions are represented on widely separated branches of the larger superfamily; how they are combined in one enzyme will be determined. In the second system, an enzyme family related to oxidoreductases functions in offloading an amide product from the carrier protein of a nonribosomal peptide synthetase. In the third system, a large family of fungal flavin-dependent monooxygenases catalyzes hydroxylation reactions on aromatic substrates in azaphilone and related pathways. The structural basis for the site- and stereo-specificity of selected family members will be determined in order to develop a panel of biocatalysts for challenging dearomatization reactions. In the fourth system, the determinants of macrocycle formation and the possibilities for an expanded substrate scope of five enzymes will be investigated.