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

Abstract

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. λ-Carrageenan is the most sulfated carrageenan and carries at least three sulfates per disaccharide unit. The sole known depolymerizing enzyme of λ-carrageenan, the λ-carrageenase from Pseudoalteromonas carrageenovora, has been purified, cloned and sequenced. Sequence analyses have revealed that the λ-carrageenase, referred to as CglA, is the first member of a new family of GHs (glycoside hydrolases), which is unrelated to families GH16, that contains κ-carrageenases, and GH82, that contains ι-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 β-propeller. The main degradation products have been purified and characterized as neo-λ-carratetraose [DP (degree of polymerization) 4] and neo-λ-carrahexaose (DP6), indicating that CglA hydrolyses the β-(1→4) linkage of λ-carrageenan. LC-MALLS (liquid chromatography-multi-angle laser light scattering) and 1H-NMR monitoring of the enzymatic degradation of λ-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-λ-carrabiose oligosaccharides, in the present study we demonstrate that the active site of CglA 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.