Degradation of {lambda}-carrageenan by Pseudoalteromonas carrageenovora {lambda}-carrageenase: a new family of glycoside hydrolases unrelated to {kappa}- and {iota}-carrageenases

Carrageenans are sulphated galactans found in the cell walls of red seaweeds. They are classified according to the number and the position of sulphate ester groups. {lambda}-Carrageenan is the most sulphated carrageenan and carries at least three sulphates per disaccharide unit. The sole known depolymerising 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 CglA, is the first member of a new family of 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 a {beta}-propeller. The main degradation products have been purified and characterized as neo-{lambda}-carratetraose (DP4) and neo-{lambda}-carrahexaose (DP6), indicating that CglA hydrolyzes the {beta}-(1->4) linkage of {lambda}-carrageenan. LC-MALLS and 1}H-NMR monitoring of the enzymatic degradation of {lambda}-carrageenan indicate that CglA proceeds according to an endolytic mode of action and a mechanism of inversion of the anomeric configuration. Using 2-aminoacridone-labelled neo-{gamma}-carrabiose oligosaccharides, we demonstrate that the active site of CglA is constituted of at least 8 subsites (-4 to +4) and that a DP6 oligosaccharide binds in the subsites -4 to +2 and can be hydrolyzed into DP4 and DP2.