Enzyme structures and analysis of the regiospecificity in natural product biosynthesis

Natural products are small chemical compounds with biological activities, which are derived from secondary metabolites of microbes, plants, or animals. Due to the in pharmacological properties, natural products have been a major source of current human medicines. The study of natural product also leads to the advancement of organic and biological chemistry. The complexity of natural product structure provides a monumental challenge for total synthesis. Study of natural product biosynthetic pathway provides diverse insight to the enzymatic mechanisms of regioselective and stereoselective reactions as well as how enzymes work with specific cofactors and coenzymes for a new template.;Recent developments in molecular biology, bioinformatics, and protein biochemistry have facilitated the characterization of natural product biosynthetic pathways and provided the alternative approach for the synthesis of small molecules. Moreover, combinational biosynthesis and modulation of synthetic pathways through protein engineering enable semisynthesis of natural product variants, which adopt structural derivatives with enhanced activity. Structural and mechanistic analysis of proteins in natural product biosynthetic pathways has guided the protein engineering for the generation of natural product variants with beneficial therapeutic properties.;The research presented in this thesis focuses on the enzyme structures in the natural product calicheamicin biosynthetic pathway and its substrate specificity expansion through the structural-based mutagenesis. Chapter 1 will provide a background of calicheamicin biosynthesis and glycosyltransferase structures and recent progress in glycosyltransferase engineering. Next two chapters describe enzyme structures in the calicheamicin biosynthesis pathway. Chapter 2 describes the structures of calicheamicin glycosyltransferases. Comparison of multiple glycosyltransferase structures in a single natural product biosynthetic pathway illustrates how proteins with low sequence homology yet similar structures mediate distinct regiospecific reactions. Chapter 3 describes the structure of calicheamicin methyltransferase, which shows a typical natural product methyltransferase structure. Chapter 4 describes the structures of nucleotidyltransfease, which is an essential enzyme for glycosyltransferase engineering. The Q83S and Q83D variants of RmlA expand nucleotide base specificity. The structures of these point mutants are presented in this chapter.