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UBC Theses and Dissertations

Studies on enzymes of sialic acid and pseudaminic acid biosynthesis Chou, Wayne Kuo Wei


The first two steps in the mammalian biosynthesis of sialic acid are catalyzed by a bifunctional hydrolyzing UDP-GlcNAc 2-epimerase/ManNAc kinase. The hydrolyzing epimerase domain, which catalyzes the conversion of UDP-GlcNAc to ManNAc and UDP, was studied mechanistically. Incubation of UDP-GlcNAc in deuterated buffer produced α-[2-²H]ManNAc indicating that the epimerase reaction proceeds with a net retention of configuration at C-1 and that C-2 is deprotonated and reprotonated with a solvent-derived deuterium atom. The enzymatic incubation of a potential reaction intermediate, 2-acetamidoglucal, resulted in a slow hydration reaction, forming ManNAc. A positional isotope exchange and kinetic isotope effect experiment demonstrated that UDP loss occurs through a C-O bond cleavage and that C-2 deprotonation is not rate-limiting. These results support a mechanism involving the anti-elimination of UDP, forming 2-acetamidoglucal, followed by its syn-hydration, forming ManNAc. SiaA, a monofunctional hydrolyzing UDP-GlcNAc 2-epimerase from Neisseria meningitidis, was recently identified to catalyze a mechanistically similar reaction to the mammalian epimerase and its study was undertaken to identify catalytic residues. Mutagenesis produced three SiaA mutants, D100N, E122Q and D131N. In each case catalysis was severely impaired, however, products were detected upon extended incubations. E122Q and D131N catalyzed the release of 2-acetamidoglucal before the formation of ManNAc, demonstrating that hydrolyzing epimerases are able to catalyze the direct formation of 2-acetamidoglucal. Pseudaminic acid synthase is proposed to catalyze the condensation of PEP and 6-deoxy AltdiNAc to form pseudaminic acid and phosphate. The activity of a pseudaminic acid synthase has never before been observed, but a gene candidate, (neuB3 from Campylobacter jejuni NCTC 11168) potentially encoding a protein fulfilling this role, was identified and cloned. The activity of NeuB3 was studied through the synthesis and enzymatic incubation of its potential substrate, 6-deoxy AltdiNAc. NMR spectroscopic analysis verified the activity of NeuB3 as a pseudaminic acid synthase and that the absolute stereochemistry of the pseudaminic acid produced had the L-glycero-L-manno configuration. Kinetic analysis revealed that NeuB3 requires a divalent metal ion for catalysis and that optimal catalysis occurs at pH 7.0. Incubation with [2-¹⁸O]PEP demonstrated that NeuB3 operates via a C-O bond cleavage mechanism utilized in PEP-condensing synthases studied to date.

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