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UBC Theses and Dissertations

Mechanistic studies of the enzymes involved in the biosynthesis of CMP-N, N'-diacetyllegionaminic acid and UDP-D-apipose Glaze, Pavel Alexander

Abstract

This thesis focuses on the biosynthesis of two sugar nucleotides. The enzymes responsible for the biosynthesis of N,N’-diacetyllegionaminic acid in Legionella pneumophila are identified for the first time. All three genes (neuA,B, C) demonstrated sequence homology to the genes involved in sialic acid biosynthesis. The first gene neuC encodes a hydrolyzing 2- epimerase, which is found to catalyze the conversion of UDP-N,N’-diacetylbacillosamine (UDP Bac2,4diNAc) into 2,4-diacetamido-2,4,6-trideoxymannose (6-deoxyMandiNAc) and uridine diphosphate (UDP). The incubation of UDP-Bac2,4diNAc with NeuC in deuterated buffer generated α-[2-²H]-6-deoxyMandiNAc. This indicates that the reaction catalyzed by the hydrolyzing 2-epimerase proceeds with a net retention of configuration at C-1, and that C-2 is deprotonated and reprotonated with a solvent-derived deuterium atom. An enzymatic reaction in H₂¹⁸0 demonstrated that the loss of UDP occurs through a C-O bond cleavage process. These results support a mechanism involving the anti-elimination of UDP, forming a 6-deoxy-2,4- diacetamidoglucal intermediate, followed by a syn-hydration, to generate 6-deoxyMandiNAc. N,N’-diacetyllegionaminic acid synthase (NeuB) is a potential phosphoenolpyruvate-condensing synthase involved in the biosynthesis of N,N’-diacetyllegionaminic acid (Leg5Ac7Ac). This enzyme is proposed to catalyze the condensation of phosphoenolpyruvate (PEP) and 6- deoxyMandiNAc to form Leg5Ac7Ac and phosphate. NMR spectroscopic analysis confirmed that NeuB is an acid synthase and that the N,N’-diacetyllegionaminic acid product has the D-glycero-D-galacto configuration. Incubation with [2-¹⁸O] ]-PEP demonstrated that NeuB operates via a C-O bond cleavage mechanism. Finally, the NeuA homolog was demonstrated to possess CMP-N,N’-diacetyllegionaminic acid synthetase activity generating CMP-Leg5Ac7Ac, which is activated for use in lipopolysaccharide biosynthesis. UDP-D-apiose is biosynthesized from UDP-D-glucuronic acid by a bifunctional enzyme UDP-D-apiose/UDP-D-xylose synthase (AXS 1). NMR spectroscopic analysis confirmed that AXS 1 produces a roughly 1:1 mixture of UDP-D-apiose (UDP-Api) and UDP-D-xylose (UDP Xyl). Incubation of a potential reaction intermediate, UDP-4-ketoxylose, resulted in the slow formation of either UDP-Xyl and possibly UDP-Api. AXS 1 catalyzed the formation of exclusively UDP-2-deoxy-2-fluoroxylose when incubated with UDP-2-deoxy-2-fluoroglucuronic acid, while fluoride, UDP and C₂O was formed when AXS1 was incubated with UDP-3-deoxy- 3-fluoroglucuronic acid. The enzymatic incubation with these substrate analogs provided further evidence for the retro-aldol mechanism.

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