The κ-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 Angstrom 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.