Identification of residues that confer sugar selectivity to UDP-glycosyltransferase 3A (UGT3A) enzymes

Meech, R., Rogers, A., Zhuang, L., Lewis, B.C., Miners, J.O. and Mackenzie, P.I. (2012) Identification of residues that confer sugar selectivity to UDP-glycosyltransferase 3A (UGT3A) enzymes. The Journal of Biological Chemistry, 287 (29), 24122-24130. (doi:10.1074/jbc.M112.343608). (PMID:22621930)

Abstract

Recent studies in this laboratory characterized the UGT3A family enzymes, UGT3A1 and UGT3A2, and showed that neither uses the traditional UGT co-substrate UDP-glucuronic acid. Rather, UGT3A1 uses N-acetylglucosamine as preferred sugar donor and UGT3A2 uses UDP-glucose. The enzymatic characterization of UGT3A mutants, structural modelling, and multispecies gene analysis have now been employed to identify a residue within the active site of these enzymes that confer their unique sugar preferences. An asparagine (N391) residue in the UGT signature sequence of UGT3A1 is necessary for utilization of UDP-N-acetylglucosamine. Conversely, a phenylalanine (F391) residue in UGT3A2 favors UDP-glucose use. Mutation of N391 to F in UGT3A1 enhances its ability to utilize UDP-glucose and completely inhibits its ability to use UDP-N-acetylglucosamine. An analysis of homology models docked with UDP-sugar donors indicates that N391 in UGT3A1 is able to accommodate the N-acetyl group on C2 of UDP N-acetylglucosamine so that the anomeric carbon atom (C1) is optimally situated for catalysis involving H35. Replacement of N by F at 391 disrupts this catalytically-productive orientation of UDP N-acetylglucosamine but allows a more optimal alignment of UDP-glucose for sugar donation. Multispecies sequence analysis reveals that only primates possess UGT3A sequences containing the N391 residue, suggesting that other mammals may not have the capacity to N-acetylglucosaminidate small molecules. In support of this hypothesis, N391-containing UGT3A forms from two non-human primates were found to use UDP-N-acetylglucosamine, while UGT3A isoforms from non-primates could not use this sugar donor. This work gives new insight into the residues that confer sugar specificity to UGT family members and suggests a primate-specific innovation in glycosidation of small molecules

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