The kappa-carrageenase of P. carrageenovora features a tunnel-shaped active site: a novel insight in the evolution of Clan-B glycoside hydrolases.
BACKGROUND
kappa-carrageenans are gel-forming, sulfated 1,3-alpha-1,4-beta-galactans from the cell walls of marine red algae. The kappa-carrageenase from the marine, gram-negative bacterium Pseudoalteromonas carrageenovora degrades kappa-carrageenan both in solution and in solid state by an endoprocessive mechanism. This beta-galactanase belongs to the clan-B of glycoside hydrolases.


RESULTS
The structure of P. carrageenovora kappa-carrageenase has been solved to 1.54 A resolution by the multiwavelength anomalous diffraction (MAD) method, using a seleno-methionine-substituted form of the enzyme. The enzyme folds into a curved beta sandwich, with a tunnel-like active site cavity. Another remarkable characteristic is the presence of an arginine residue at subsite -1.


CONCLUSIONS
The crystal structure of P. carrageenovora kappa-carrageenase is the first three-dimensional structure of a carrageenase. Its tunnel-shaped active site, the first to be reported for enzymes other than cellulases, suggests that such tunnels are associated with the degradation of solid polysaccharides. Clan-B glycoside hydrolases fall into two subgroups, one with catalytic machinery held by an ancestral beta bulge, and the other in which it is held by a regular beta strand. At subsite -1, all of these hydrolases exhibit an aromatic amino acid that interacts with the hexopyranose ring of the monosaccharide undergoing catalysis. In addition, in kappa-carrageenases, an arginine residue recognizes the sulfate-ester substituents of the beta-linked kappa-carrageenan monomers. It also appears that, in addition to the nucleophile and acid/base catalysts, two other amino acids are involved with the catalytic cycle, accelerating the deglycosylation step.
Michel
G
G
Laboratoire de Cristallographie Macromoléculaire, Institut de Biologie Structurale Jean-Pierre Ebel, CNRS/CEA, 41 Avenue des Martyrs, 38027 Cedex 1, Grenoble, France.
Chantalat
L
L
Duee
E
E
Barbeyron
T
T
Henrissat
B
B
Kloareg
B
B
Dideberg
O
O
D020591:Alteromonas / Q000201:enzymology*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D002240:Carbohydrate Sequence; D019143:Evolution, Molecular; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D006026:Glycoside Hydrolases; C019846:kappa-carrageenase protein, Alteromonadaceae