Complex and unique enzymology is often behind the biosynthesis of natural products. This thesis is focused on how non-proteinogenic amino acids are biosynthesized and then incorporated into natural products. Chapters two, three and four deal with a unique dioxygenase found in vancomycin biosynthesis. Chapter five elaborates on the biochemical characterization along with efforts toward structural characterization of a terminal non-ribosomal peptide synthetase module.
The vancomycin biosynthetic enzyme DpgC belongs to a small class of oxygenation enzymes that are not dependent on an accessory cofactor or metal ion. The detailed mechanism of cofactor-independent oxygenases has not been established. We have solved the first structure of an enzyme of this oxygenase class complexed with a bound substrate mimic. The use of a designed, synthetic substrate analog allows unique insights into the chemistry of oxygen activation. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is present adjacent to the site of oxidation on the substrate. Molecular oxygen is bound in a small hydrophobic pocket and the substrate provides the reducing power to activate oxygen for downstream chemical steps. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics. Mechanistic parallels exist between DpgC and cofactor-dependent flavoenzymes, providing information regarding the general mechanism of enzymatic oxygen activation.
