title: 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 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.
authors.affiliation: Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway.
authors.forename: Tonje M
authors.initials: TM
authors.lastname: Bjerkan
authors.forename: Carol L
authors.initials: CL
authors.lastname: Bender
authors.forename: Helga
authors.initials: H
authors.lastname: Ertesvåg
authors.forename: Finn
authors.initials: F
authors.lastname: Drabløs
authors.forename: Mohamed K
authors.initials: MK
authors.lastname: Fakhr
authors.forename: Lori A
authors.initials: LA
authors.lastname: Preston
authors.forename: Gudmund
authors.initials: G
authors.lastname: Skjak-Braek
authors.forename: Svein
authors.initials: S
authors.lastname: Valla
mesh_terms: D000464:Alginates / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D005782:Gels / Q000737:chemistry / Q000378:metabolism; D016680:Genome, Bacterial; D006867:Hydrolases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010802:Phylogeny; D044224:Pseudomonas syringae / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D000464:Alginates; D001426:Bacterial Proteins; D005782:Gels; D006867:Hydrolases; D002238:Carbohydrate Epimerases; D002118:Calcium