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

Abstract

Alginates are industrially important, linear copolymers of β-D-mannuronic acid (M) and its C-5-epimer α-L-guluronic acid (G). The G residues originate from a post-polymerization 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 Ca2+ -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 PsmEA activity. PsmE contains two regions designated M and RTX, both presumably involved in the binding of Ca2+. 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 on of N and A PsmE was capable of redesigning an extensively acetylated alginate low in G from a non gel-forming to a gel-forminging state. Such a property has to our kmowledge not been previously reported for an enzyme acting on a polysaccharide.