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Identification and mechanism of a bacterial hydrolyzing UDP-N-acetylglucosamine 2-epimerase

TitleIdentification and mechanism of a bacterial hydrolyzing UDP-N-acetylglucosamine 2-epimerase
Publication TypeJournal Article
Year of Publication2004
AuthorsMurkin, AS, Chou, WK, Wakarchuk, WW, Tanner, ME
JournalBiochemistry
Volume43
Pagination14290-14298
Date PublishedNov
Type of ArticleArticle
ISBN Number0006-2960
Keywords2-EPIMERASE/N-ACETYLMANNOSAMINE, biosynthesis, CELL-INTERACTIONS, ENZYME, ESCHERICHIA-COLI K1, KINASE, POLYSIALIC ACID, PURIFICATION, RAT-LIVER, SIALIC-ACID, STEREOCHEMISTRY
Abstract

This paper reports the first identification of a fully functional hydrolyzing UDP-N-acetylglucosamine 2-epimerase from a bacterial source. The epimerase (known as SiaA or NeuC from Neisseria meningitidis MC58 group B is shown to catalyze the conversion of UDP-GlcNAc into ManNAc and UDP in the first step of sialic acid (N-acetylneuraminic acid) biosynthesis. The mechanism is proposed to involve an anti elimination of UDP to form 2-acetamidoglucal as an intermediate, followed by the syn addition of water. The observation that the alpha-anomer of ManNAc is the true product and that solvent deuterium is incorporated at C-2 is consistent with this mechanism. The use of the O-18-labeled substrate confirms that the overall hydrolysis reaction proceeds via cleavage of the C-O bond. Furthermore, the putative intermediate 2-acetamidoglucal is shown to serve as a catalytically competent substrate and is enzymatically hydrated to give ManNAc exclusively. Isotope effect studies show that cleavage of the C-H bond is not rate limiting during catalysis. Mutagenesis studies show that three active site carboxylate residues are crucial for catalysis. In two of the mutants that were studied (E122Q and D131N), 2-acetamidoglucal was released from the active site during catalysis, providing direct evidence that the enzyme is capable of catalyzing the anti elimination of UDP from UDP-GlcNAc.

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