| GDP-colitose and GDP-perosamine are deoxysugars and are constituents of the O-antigens found within various Gram negative bacterial species including Escherichia coli, Vibrio cholerae, and Salmonella enterica. Both sugars are derived from GDP-mannose, which first undergoes an oxidation/reduction reaction by GDP-mannose-4,6-dehydratase to produce GDP-4-keto-6-deoxymannose. ColD removes the 3'-OH group from the hexose moiety of this intermediate, after which additional reactions produce GDP-colitose. GDP-perosamine synthase is an aminotransferase that also acts upon GDP-4-keto-6-deoxymannose, but instead converts the 4'-keto group into an amino group, thereby producing GDP-perosamine. ColD and GDP-perosamine synthase are both pyridoxal-5'-phosphate (PLP)-dependent enzymes, utilize the same substrates, and share an amino acid sequence identity of 23%. Presented in this dissertation is a combined structural and functional study of these two enzymes.;Wild-type structures of both ColD from E. coli O55 and GDP-perosamine synthase from Caulobacter crescentus CB 15 demonstrate that these enzymes belong to the well-characterized aspartate aminotransferase family of PLP-dependent enzymes. Crystal packing interactions and subsequent gel filtration experiments suggest that both enzymes are dimers in their active forms. Examination of the active sites suggests that His 188 and Lys 186 act as the sole general acid/base in ColD and GDP-perosamine synthase, respectively. Site-directed mutant enzyme structures with trapped sugar compounds demonstrate the manner in which these enzymes accommodate their sugar substrates in the active site. On the basis of these structures, a site-directed mutant ColD enzyme was constructed that is no longer a 3-dehydratase, but is instead a 4-aminotransferase. Additionally, by utilizing the wild-type ColD product (GDP-4-keto-3,6-dideoxymannose) as an alternative substrate for GDP-perosamine synthase and subsequently GDP-perosamine acetyltransferase, two novel nucleotide-linked sugar compounds were produced. The ability to alter the function of an enzyme through site-directed mutagenesis and to produce new sugar compounds, as demonstrated in this dissertation, is useful in generating novel sugar-containing therapeutic natural products from existing scaffolds. |
