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Biochemistry. 1997 Jan 28;36(4):796-805.

Kinetic characterization of wild-type and S229A mutant MurB: evidence for the role of Ser 229 as a general acid.

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  • 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.

Abstract

X-ray derived structural data predicted that serine 229 was positioned to act as a proton donor to the developing C2 carbanion during the reduction of enolpyruvyl-UDP-N-acetylglucosamine catalyzed by the bacterial peptidoglycan biosynthetic flavoenzyme MurB. To investigate this effect, a mutant where serine 229 was replaced by alanine was constructed and purified. Kinetic analysis of the two half-reactions for the mutant enzyme revealed a 9-fold decrease in the reduction of EFlox by NADPH and a dramatic 10(7)-fold decrease in the reoxidation of EFlred with the enolpyruvyl substrate. In addition, studies of S229A with the substrate analog, (E)-enolbutyryl-UDP-N-acetylglucosamine, showed a striking bias of the partitioning toward formation of the (Z) geometric isomer as opposed to formation of the reduced product UDP-methylmuramic acid, which was the predominant product in wild-type MurB. These studies provide evidence for the proposed role of this active-site serine as a general acid catalyst.

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