title: Insights into role of the hydrogen bond networks in substrate recognition by UDP-GalNAc 4-epimerases.
abstract: UDP-hexose 4-epimerases are critical in galactose metabolism and often important in lipopolysaccharide biosynthesis as well. Three groups of these enzymes have been reported based on their substrate specificity towards non-acetylated substrates (group 1), dual specificity towards N-acetylated and non-acetylated substrates (group 2) and specificity towards N-acetylated substrates (group 3). We recently reported the structure of a novel UDP-GalNAc 4-epimerase called WbgU and based on the structure proposed a model of specific substrate recognition by UDP-GalNAc 4-epimerases. In this work, we present an analysis of the proposed model of substrate recognition using site-directed mutagenesis of WbgU and crystal structure of the His305Ala mutant. This investigation reveals that the wild-type activity of WbgU is retained in most single-point mutants targeting the active site. However, a graded loss in activity is observed in double-and triple-point mutants with the quadruple-point mutant being completely inactive corroborating the proposed rationale of substrate recognition. Furthermore, crystal structure of the His305Ala mutant shows that the structure is significantly similar to the wild-type WbgU, albeit a loss in the critical hydrogen bond network seated at His305 and ensuing minor conformational changes. It is inferred that the specific and non-specific interactions throughout the active site confer it sufficient elasticity to sustain wild-type activity for several of the single-point mutations.
authors.affiliation: Department of Biochemistry, The Ohio State University, Columbus, OH 43210, United States.
authors.forename: Veer Sandeep
authors.initials: VS
authors.lastname: Bhatt
authors.forename: Wanyi
authors.initials: W
authors.lastname: Guan
authors.forename: Mengyang
authors.initials: M
authors.lastname: Xue
authors.forename: Huiqing
authors.initials: H
authors.lastname: Yuan
authors.forename: Peng George
authors.initials: PG
authors.lastname: Wang
mesh_terms: D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D002238:Carbohydrate Epimerases; C559984:UDP-N-acetylgalactosamine 4-epimerase, Pleisomonas shigelloides