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-  2019 

UDP-glucose Dehydrogenase: The First-step Oxidation Is an NAD+-dependent Bimolecular Nucleophilic Substitution Reaction (SN2)

DOI: 10.7150/ijbs.28904

Keywords: bimolecular nucleophilic substitution reaction (SN2), catalytic mechanism, NAD+-dependent, two-fold oxidation, UDP-glucose dehydrogenase

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Abstract:

UDP-glucose dehydrogenase (UGDH) catalyzes the conversion of UDP-glucose to UDP-glucuronic acid by NAD+-dependent two-fold oxidation. Despite extensive investigation into the catalytic mechanism of UGDH, the previously proposed mechanisms regarding the first-step oxidation are somewhat controversial and inconsistent with some biochemical evidence, which instead supports a mechanism involving an NAD+-dependent bimolecular nucleophilic substitution (SN2) reaction. To verify this speculation, the essential Cys residue of Streptococcus zooepidemicus UGDH (SzUGDH) was changed to an Ala residue, and the resulting Cys260Ala mutant and SzUGDH were then co-expressed in vivo via a single-crossover homologous recombination method. Contrary to the previously proposed mechanisms, which predict the formation of the capsular polysaccharide hyaluronan, the resulting strain instead produced an amide derivative of hyaluronan, as validated via proteinase K digestion, ninhydrin reaction, FT-IR and NMR. This result is compatible with the NAD+-dependent SN2 mechanism.

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