Characterization of mannuronan C-5-epimerase genes from the brown alga Laminaria digitata.
Alginate is an industrially important polysaccharide obtained commercially by harvesting brown algae. The final step in alginate biosynthesis, the epimerization of beta-1,4-d-mannuronic acid to alpha-1,4-l-guluronic acid, a structural change that controls the physicochemical properties of the alginate, is catalyzed by the enzyme mannuronan C-5-epimerase. Six different cDNAs with homology to bacterial mannuronan C-5-epimerases were isolated from the brown alga Laminaria digitata (Phaeophyceae). Hydrophobic cluster analysis indicated that the proteins encoded by the L. digitata sequences have important structural similarities to the bacterial mannuronan C-5-epimerases, including conservation of the catalytic site. The expression of the C-5-epimerase genes was examined by northern-blot analysis and reverse transcriptase-polymerase chain reaction in L. digitata throughout a year. Expression was also monitored in protoplast cultures by northern and western blot, reverse transcriptase-polymerase chain reaction, and activity measurements. From both the structural comparisons and the expression pattern, it appears that the cDNAs isolated from L. digitata encode functional mannuronan C-5-epimerases. The phylogenetic relationships of the bacterial and brown algal enzymes and the inferences on the origin of alginate biosynthetic machinery are discussed.
Nyvall
Pi
P
Unité Mixte de Recherche 1931, Centre National de la Recherche Scientifique and Laboratoires Goëmar, Station Biologique de Roscoff, BP 74, 29682 Roscoff cedex, Brittany, France.
Corre
Erwan
E
Boisset
Claire
C
Barbeyron
Tristan
T
Rousvoal
Sylvie
S
Scornet
Delphine
D
Kloareg
Bernard
B
Boyen
Catherine
C
D000595:Amino Acid Sequence; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000145:classification / Q000235:genetics*; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D017343:Genes, Plant; D007795:Laminaria / Q000145:classification / Q000201:enzymology / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D010802:Phylogeny; D011487:Protein Conformation; D011523:Protoplasts / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D002238:Carbohydrate Epimerases