Degradation of λ-carrageenan by Pseudoalteromonas carrageenovora λ-carrageenase:: a new family of glycoside hydrolases unrelated to κ- and ι-carrageenases

Carrageenans are sulfated galactans found in the cell walls of red seaweeds. They are classified according to the number and the position of sulfate ester groups. lambda-Carrageenan is the most sulfated carrageenan and carries at least three sulfates per disaccharide unit. The sole known depolymerizing enzyme of lambda-carrageenan, the; lambda-carrageenase from Pseudoalteromonas carrageenovora, has been purified, cloned and sequenced. Sequence analyses have revealed that the lambda-carrageenase, referred to as Cg1A, is the first member of a new family of GHs (glycoside hydrolases), which is unrelated to families GH16, that contains kappa-carrageenases, and GH82, that contains iota-carrageenases. This large enzyme (105 kDa) features a low-complexity region, suggesting the presence of a linker connecting at least two independent modules. The N-terminal region is predicted to fold as beta-propeller. The main degradation products have been purified and characterized as neo-lambda-carratetraose [DP (degree of polymerization) 4] and neo-lambda-carrahexaose (DP6), indicating that Cg1A hydrolyses the beta-(1 -> 4) linkage of lambda-carrageenan. LC-MALLS (liquid chromatography-multi-angle laser light scattering) and H-1-NMR monitoring of the enzymatic degradation of lambda-carrageenan indicate that Cg1A proceeds according to an endolytic mode of action and a mechanism of inversion of the anomeric configuration. Using 2-aminoacridone-labelled neo-lambda-carrabiose oligosaccharides, in the present study we demonstrate that the active site of Cg1A comprises at least 8 subsites (-4 to +4) and that a DP6 oligosaccharide binds in the subsites -4 to +2 and can be hydrolysed into DP4 and DP2.