Polysaccharide synthesis of the levansucrase SacB from Bacillus megaterium is controlled by distinct surface motifs

J Biol Chem. 2011 May 20;286(20):17593-600. doi: 10.1074/jbc.M110.203166. Epub 2011 Mar 25.

Abstract

Despite the widespread biological function of carbohydrates, the polysaccharide synthesis mechanisms of glycosyltransferases remain largely unexplored. Bacterial levansucrases (glycoside hydrolase family 68) synthesize high molecular weight, β-(2,6)-linked levan from sucrose by transfer of fructosyl units. The kinetic and biochemical characterization of Bacillus megaterium levansucrase SacB variants Y247A, Y247W, N252A, D257A, and K373A reveal novel surface motifs remote from the sucrose binding site with distinct influence on the polysaccharide product spectrum. The wild type activity (k(cat)) and substrate affinity (K(m)) are maintained. The structures of the SacB variants reveal clearly distinguishable subsites for polysaccharide synthesis as well as an intact active site architecture. These results lead to a new understanding of polysaccharide synthesis mechanisms. The identified surface motifs are discussed in the context of related glycosyltransferases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Substitution
  • Bacillus megaterium / enzymology*
  • Bacillus megaterium / genetics
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Hexosyltransferases / chemistry
  • Hexosyltransferases / genetics
  • Hexosyltransferases / metabolism*
  • Mutation, Missense
  • Polysaccharides, Bacterial / biosynthesis*
  • Polysaccharides, Bacterial / genetics

Substances

  • Bacterial Proteins
  • Polysaccharides, Bacterial
  • Hexosyltransferases
  • levansucrase

Associated data

  • PDB/3OM2
  • PDB/3OM4
  • PDB/3OM5
  • PDB/3OM6
  • PDB/3OM7