Abstract
Carrageenans are gel-forming hydrocolloids extracted from the cell walls of marine red algae. They consist of d-galactose residues bound by alternate alpha(1-->3) and beta(1-->4) linkages and substituted by one (kappa-carrageenan), two (iota-carrageenan), or three (lambda-carrageenan) sulfate-ester groups per disaccharide repeating unit. Both the kappa- and iota-carrageenan chains adopt ordered conformations leading to the formation of highly ordered aggregates of double-stranded helices. Several kappa-carrageenases and iota-carrageenases have been cloned from marine bacteria. Kappa-carrageenases belong to family 16 of the glycoside hydrolases, which essentially encompasses polysaccharidases specialized in the hydrolysis of the neutral polysaccharides such as agarose, laminarin, lichenan, and xyloglucan. In contrast, iota-carrageenases constitute a novel glycoside hydrolase structural family. We report here the crystal structure of Alteromonas fortis iota-carrageenase at 1.6 A resolution. The enzyme folds into a right-handed parallel beta-helix of 10 complete turns with two additional C-terminal domains. Glu(245), Asp(247), or Glu(310), in the cleft of the enzyme, are proposed as candidate catalytic residues. The protein contains one sodium and one chloride binding site and three calcium binding sites shown to be involved in stabilizing the enzyme structure.
Highlights
Carrageenans are gel-forming hydrocolloids extracted from the cell walls of marine red algae
Several -carrageenases and -carrageenases have been cloned from marine bacteria. -Carrageenases belong to family 16 of the glycoside hydrolases, which essentially encompasses polysaccharidases specialized in the hydrolysis of the neutral polysaccharides such as agarose, laminarin, lichenan, and xyloglucan
Carrageenans are the main components of the cell walls of various marine red algae (Rhodophyta) where they play a variety of structural and signaling roles [1, 2]
Summary
Expression, Purification, and Crystallization of Native and Selenomethionyl--carrageenase—Full details of the expression and purification of native -carrageenase have been described previously [15]. The Se-Met--carrageenase appeared to be less soluble than the native protein and could only be concentrated to 2 mg/ml. Crystallization of Se-Met--carrageenase was performed under similar conditions to those already reported [15] except for the addition of 1 mM dithiothreitol and the replacement of sodium cacodylate by imidazole to avoid the reaction between cacodylate and dithiothreitol [17]. Under these conditions, small crystals with a maximal size of 100 ϫ 100 ϫ 50 m were obtained in 1 month of equilibration, whereas large native crystals appeared in a few days.
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