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Mechanistic studies and manipulation of the enzymes in sialic acid and pseudaminic acid biosynthesis Liu, Feng


The Neisseria meningitidis sialic acid synthase (NeuB) catalyzes the metal-dependent condensation of N-acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP) to generate N-acetylneuraminic acid. This work describes the synthesis and characterization of the first potent inhibitor of sialic acid synthase, a tetrahedral intermediate analogue as a mixture of stereoisomers at the key tetrahedral center. Inhibition studies demonstrate that one stereoisomer binds more tightly than the other. An X-ray crystallographic analysis of the NeuB•inhibitor•Mn²⁺ complex solved to a resolution of 1.75 Å shows that the more tightly bound stereoisomer bears a (2R)-configuration. This suggests that the tetrahedral intermediate formed in the NeuB reaction also bears a (2R)-configuration. This analysis demonstrates that the active site metal serves as a source of nucleophilic water and delivers it to the si face of the oxocarbenium intermediate to generate a tetrahedral intermediate with a (2R)-configuration. The flagellin proteins in pathogenic bacteria such as Campylobacter jejuni and Helicobacter pylori are heavily glycosylated with the nine-carbon α-keto acid, pseudaminic acid. A key step in pseudaminic acid biosynthesis has been shown to involve the generation of 6-deoxy-AltdiNAc from its nucleotide-linked form, UDP-6-deoxy-AltdiNAc, by the action of a hydrolase that cleaves the glycosidic bond and releases UDP. This thesis describes the first characterization of a UDP-6-deoxy-AltdiNAc hydrolase, namely PseG (Cj1312) from C. jejuni. The activity of this enzyme is independent of the presence of divalent metal ions, and the values of the catalytic constants were found to be kcat = 27 s⁻­­­­­¹ and KM = 174 µM. The enzyme was shown to hydrolyze the substrate with an overall inversion of stereochemistry at C1 and to utilize a C-O bond cleavage mechanism during catalysis. The last part of the thesis describes the engineering of C. jejuni. We demonstrated that by feeding non-motile mutant C. jejuni bacteria with a neutral azide-labeled pseudaminic acid precursor, the mutants regained the ability to generate functional azido-bearing flagella and their motility was restored. The presence of the azido-pseudaminic acid on the surface of the flagella provides a bioorthogonal chemical handle that can be used to modify the flagellar proteins and to engineer bacteria for further studies.

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