The Pseudomonas fluorescens AlgG protein, but not its mannuronan C-5-epimerase activity, is needed for alginate polymer formation.
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.
Gimmestad
Martin
M
Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway.
Sletta
Håvard
H
Ertesvåg
Helga
H
Bakkevig
Karianne
K
Jain
Sumita
S
Suh
Sang-jin
SJ
Skjåk-Braek
Gudmund
G
Ellingsen
Trond E
TE
Ohman
Dennis E
DE
Valla
Svein
S
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
D000464:Alginates; D006603:Hexuronic Acids; D011108:Polymers; D020723:Glucuronic Acid; C008324:mannuronic acid; D002238:Carbohydrate Epimerases; C086519:mannuronan c-5-epimerase