title: Identification of residues that confer sugar selectivity to UDP-glycosyltransferase 3A (UGT3A) enzymes.
abstract: Recent studies in this laboratory characterized the UGT3A family enzymes, UGT3A1 and UGT3A2, and showed that neither uses the traditional UDP-glycosyltransferase UGT co-substrate UDP-glucuronic acid. Rather, UGT3A1 uses GlcNAc as preferred sugar donor and UGT3A2 uses UDP-Glc. The enzymatic characterization of UGT3A mutants, structural modeling, and multispecies gene analysis have now been employed to identify a residue within the active site of these enzymes that confers their unique sugar preferences. An asparagine (Asn-391) in the UGT signature sequence of UGT3A1 is necessary for utilization of UDP-GlcNAc. Conversely, a phenylalanine (Phe-391) in UGT3A2 favors UDP-Glc use. Mutation of Asn-391 to Phe in UGT3A1 enhances its ability to utilize UDP-Glc and completely inhibits its ability to use UDP-GlcNAc. An analysis of homology models docked with UDP-sugar donors indicates that Asn-391 in UGT3A1 is able to accommodate the N-acetyl group on C2 of UDP-GlcNAc so that the anomeric carbon atom (C1) is optimally situated for catalysis involving His-35. Replacement of Asn with Phe at position 391 disrupts this catalytically productive orientation of UDP-GlcNAc but allows a more optimal alignment of UDP-Glc for sugar donation. Multispecies sequence analysis reveals that only primates possess UGT3A sequences containing Asn-391, suggesting that other mammals may not have the capacity to N-acetylglucosaminidate small molecules. In support of this hypothesis, Asn-391-containing UGT3A forms from two non-human primates were found to use UDP-GlcNAc, whereas 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.
authors.affiliation: Department of Clinical Pharmacology, Flinders University School of Medicine and Flinders Medical Centre, Bedford Park, South Australia 5042, Australia. robyn.meech@flinders.edu.au
authors.forename: Robyn
authors.initials: R
authors.lastname: Meech
authors.forename: Anne
authors.initials: A
authors.lastname: Rogers
authors.forename: Lizhe
authors.initials: L
authors.lastname: Zhuang
authors.forename: Benjamin C
authors.initials: BC
authors.lastname: Lewis
authors.forename: John O
authors.initials: JO
authors.lastname: Miners
authors.forename: Peter I
authors.initials: PI
authors.lastname: Mackenzie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D020134:Catalytic Domain; D002417:Cattle; D002460:Cell Line; D019833:Genistein / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
chemical_list: D014539:Uridine Diphosphate Sugars; D019833:Genistein; D016695:Glycosyltransferases; C557504:UDP glycosyltransferase 3A2, human; D017351:N-Acetylglucosaminyltransferases; C538791:UGT3A1 protein, human; D014453:Glucuronosyltransferase