Author ORCID Identifier

https://orcid.org/0000-0002-2956-9781

Document Type

Article

Publication Date

6-2010

Publication Title

Canadian Journal of Chemistry

Volume

88

Issue

8

First Page

804

Last Page

814

DOI

10.1139/V10-044

Keywords

density functional theory (DFT), uridine diphosphate glucose dehydrogenase (UDPGlcDH), NAD, thioester hydrolysis, acid–base, electron transfer, proton transfer

Abstract

Uridine 5′-diphosphate glucuronic acid (UDPGlcUA) is a key intermediary metabolite in many species, including pathogenic bacteria and humans. It is biosynthesized from UDP-glucose (UDPGlc) by uridine diphosphate glucose dehydrogenase (UDPGlcDH) via a twofold two-electron–one-proton oxidation that successively transforms the 6-hydroxymethyl of glucopyranose into a formyl, and the latter into the final carboxylic function. The catalytic mechanism of UDPGlcDH was investigated using a large enzyme active-site model in combination with the B3LYP method and the polarizable continuum model (IEF-PCM) self-consistent reaction field. The latter was used to correct for the long-range electrostatic effect of the protein environment. The overall mechanism consists of four catalytic steps: (i) NAD+-dependent oxidation of glucose to glucuronaldehyde, (ii) nucleophilic addition of Cys260–SH to glucuronaldehyde to form a 6-thiohemiacetal intermediate, (iii) NAD+-dependent oxidation of the 6-thiohemiacetal to form a 6-thioester intermediate, and finally, (iv) hydrolysis of the 6-thioester to give glucuronic acid. In addition, this study also provides insight into the debated roles of Lys204 and Asp264, and the most likely protonation state of a reactive Michaelis complex of UDPGlcDH.

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