title: The Pseudomonas fluorescens AlgG protein, but not its mannuronan C-5-epimerase activity, is needed for alginate polymer formation.
abstract: Bacterial alginates are produced as 1-4-linked beta-D-mannuronan, followed by epimerization of some of the mannuronic acid residues to alpha-L-guluronic acid. Here we report the isolation of four different epimerization-defective point mutants of the periplasmic Pseudomonas fluorescens mannuronan C-5-epimerase AlgG. All mutations affected amino acids conserved among AlgG-epimerases and were clustered in a part of the enzyme also sharing some sequence similarity to a group of secreted epimerases previously reported in Azotobacter vinelandii. An algG-deletion mutant was constructed and found to produce predominantly a dimer containing a 4-deoxy-L-erythro-hex-4-enepyranosyluronate residue at the nonreducing end and a mannuronic acid residue at the reducing end. The production of this dimer is the result of the activity of an alginate lyase, AlgL, whose in vivo activity is much more limited in the presence of AlgG. A strain expressing both an epimerase-defective (point mutation) and a wild-type epimerase was constructed and shown to produce two types of alginate molecules: one class being pure mannuronan and the other having the wild-type content of guluronic acid residues. This formation of two distinct classes of polymers in a genetically pure cell line can be explained by assuming that AlgG is part of a periplasmic protein complex.
authors.affiliation: Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Martin
authors.initials: M
authors.lastname: Gimmestad
authors.forename: Håvard
authors.initials: H
authors.lastname: Sletta
authors.forename: Helga
authors.initials: H
authors.lastname: Ertesvåg
authors.forename: Karianne
authors.initials: K
authors.lastname: Bakkevig
authors.forename: Sumita
authors.initials: S
authors.lastname: Jain
authors.forename: Sang-jin
authors.initials: SJ
authors.lastname: Suh
authors.forename: Gudmund
authors.initials: G
authors.lastname: Skjåk-Braek
authors.forename: Trond E
authors.initials: TE
authors.lastname: Ellingsen
authors.forename: Dennis E
authors.initials: DE
authors.lastname: Ohman
authors.forename: Svein
authors.initials: S
authors.lastname: Valla
mesh_terms: D000464:Alginates / Q000378:metabolism*; D000595:Amino Acid Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D008969:Molecular Sequence Data; D017354:Point Mutation; D011108:Polymers / Q000378:metabolism*; D011551:Pseudomonas fluorescens / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D000464:Alginates; D006603:Hexuronic Acids; D011108:Polymers; D020723:Glucuronic Acid; C008324:mannuronic acid; D002238:Carbohydrate Epimerases; C086519:mannuronan c-5-epimerase