The Pseudomonas syringae genome encodes a combined mannuronan C-5-epimerase and O-acetylhydrolase, which strongly enhances the predicted gel-forming properties of alginates

J Biol Chem. 2004 Jul 9;279(28):28920-9. doi: 10.1074/jbc.M313293200. Epub 2004 Apr 27.

Abstract

Alginates are industrially important, linear copolymers of beta-d-mannuronic acid (M) and its C-5-epimer alpha-l-guluronic acid (G). The G residues originate from a postpolymerization reaction catalyzed by mannuronan C-5-epimerases (MEs), leading to extensive variability in M/G ratios and distribution patterns. Alginates containing long continuous stretches of G residues (G blocks) can form strong gels, a polymer type not found in alginate-producing bacteria belonging to the genus Pseudomonas. Here we show that the Pseudomonas syringae genome encodes a Ca(2+)-dependent ME (PsmE) that efficiently forms such G blocks in vitro. The deduced PsmE protein consists of 1610 amino acids and is a modular enzyme related to the previously characterized family of Azotobacter vinelandii ME (AlgE1-7). A- and R-like modules with sequence similarity to those in the AlgE enzymes are found in PsmE, and the A module of PsmE (PsmEA) was found to be sufficient for epimerization. Interestingly, an R module from AlgE4 stimulated Ps-mEA activity. PsmE contains two regions designated M and RTX, both presumably involved in the binding of Ca(2+). Bacterial alginates are partly acetylated, and such modified residues cannot be epimerized. Based on a detailed computer-assisted analysis and experimental studies another PsmE region, designated N, was found to encode an acetylhydrolase. By the combined action of N and A PsmE was capable of redesigning an extensively acetylated alginate low in G from a non gel-forming to a gel-forming state. Such a property has to our knowledge not been previously reported for an enzyme acting on a polysaccharide.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alginates / chemistry
  • Alginates / metabolism*
  • Amino Acid Sequence
  • Bacterial Proteins / classification
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Calcium / metabolism
  • Carbohydrate Epimerases / classification
  • Carbohydrate Epimerases / genetics
  • Carbohydrate Epimerases / metabolism*
  • Gels / chemistry
  • Gels / metabolism
  • Genome, Bacterial
  • Hydrolases / classification
  • Hydrolases / genetics
  • Hydrolases / metabolism*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Nuclear Magnetic Resonance, Biomolecular
  • Phylogeny
  • Pseudomonas syringae / enzymology*
  • Pseudomonas syringae / genetics
  • Sequence Alignment

Substances

  • Alginates
  • Bacterial Proteins
  • Gels
  • Hydrolases
  • Carbohydrate Epimerases
  • Calcium

Associated data

  • GENBANK/AY493437