Identification and mechanism of a bacterial hydrolyzing UDP-N-acetylglucosamine 2-epimerase.
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 (18)O-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.
Murkin
Andrew S
AS
Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1.
Chou
Wayne K
WK
Wakarchuk
Warren W
WW
Tanner
Martin E
ME
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001665:Binding Sites / Q000235:genetics; D002238:Carbohydrate Epimerases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002384:Catalysis; D003001:Cloning, Molecular; D041961:Deuterium Exchange Measurement; D015964:Gene Expression Regulation, Bacterial; D006595:Hexosamines / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009345:Neisseria meningitidis / Q000201:enzymology* / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011817:Rabbits; D019998:Racemases and Epimerases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012997:Solvents; D013237:Stereoisomerism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D006595:Hexosamines; D012997:Solvents; C019231:2-acetamidoglucal; D019998:Racemases and Epimerases; D002238:Carbohydrate Epimerases; C091606:NeuC protein, Neisseria meningitidis; C100033:UDP acetylglucosamine-2-epimerase