Structure/function analysis of Pasteurella multocida heparosan synthases: toward defining enzyme specificity and engineering novel catalysts

J Biol Chem. 2012 Mar 2;287(10):7203-12. doi: 10.1074/jbc.M111.311704. Epub 2012 Jan 10.

Abstract

The Pasteurella multocida heparosan synthases, PmHS1 and PmHS2, are homologous (∼65% identical) bifunctional glycosyltransferase proteins found in Type D Pasteurella. These unique enzymes are able to generate the glycosaminoglycan heparosan by polymerizing sugars to form repeating disaccharide units from the donor molecules UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc). Although these isozymes both generate heparosan, the catalytic phenotypes of these isozymes are quite different. Specifically, during in vitro synthesis, PmHS2 is better able to generate polysaccharide in the absence of exogenous acceptor (de novo synthesis) than PmHS1. Additionally, each of these enzymes is able to generate polysaccharide using unnatural sugar analogs in vitro, but they exhibit differences in the substitution patterns of the analogs they will employ. A series of chimeric enzymes has been generated consisting of various portions of both of the Pasteurella heparosan synthases in a single polypeptide chain. In vitro radiochemical sugar incorporation assays using these purified chimeric enzymes have shown that most of the constructs are enzymatically active, and some possess novel characteristics including the ability to produce nearly monodisperse polysaccharides with an expanded range of sugar analogs. Comparison of the kinetic properties and the sequences of the wild-type enzymes with the chimeric enzymes has enabled us to identify regions that may be responsible for some aspects of both donor binding specificity and acceptor usage. In combination with previous work, these approaches have enabled us to better understand the structure/function relationship of this unique family of glycosyltransferases.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Glycosyltransferases / chemistry*
  • Glycosyltransferases / genetics
  • Glycosyltransferases / metabolism
  • Isoenzymes / chemistry
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Pasteurella multocida / enzymology*
  • Pasteurella multocida / genetics
  • Polysaccharides, Bacterial / biosynthesis
  • Polysaccharides, Bacterial / chemistry
  • Polysaccharides, Bacterial / genetics
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Structure-Activity Relationship
  • Uridine Diphosphate Glucuronic Acid / chemistry
  • Uridine Diphosphate Glucuronic Acid / genetics
  • Uridine Diphosphate Glucuronic Acid / metabolism
  • Uridine Diphosphate N-Acetylglucosamine / chemistry
  • Uridine Diphosphate N-Acetylglucosamine / genetics
  • Uridine Diphosphate N-Acetylglucosamine / metabolism

Substances

  • Bacterial Proteins
  • Isoenzymes
  • Polysaccharides, Bacterial
  • Recombinant Fusion Proteins
  • Uridine Diphosphate Glucuronic Acid
  • Uridine Diphosphate N-Acetylglucosamine
  • Glycosyltransferases
  • heparosan synthase