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Cell walls of marine macroalgae are composed of diverse polysaccharides that provide abundant carbon sources for marine heterotrophic bacteria. Among them, Zobellia galactanivorans is considered as a model for studying algae–bacteria interactions. The degradation of typical algal polysaccharides, such as agars or alginate, has been intensively studied in this model bacterium, but the catabolism of plant-like polysaccharides is essentially uncharacterized. Here, we identify a polysaccharide utilization locus in the genome of Z. galactanivorans, induced by laminarin (β-1,3-glucans), and containing a putative GH5 subfamily 4 (GH5_4) enzyme, currently annotated as a endoglucanase (ZgEngAGH5_4). A phylogenetic analysis indicates that ZgEngAGH5_4 was laterally acquired from an ancestral Actinobacteria. We performed the biochemical and structural characterization of ZgEngAGH5_4 and demonstrated that this GH5 is, in fact, an endo-β-glucanase, most active on mixed-linked glucan (MLG). Although ZgEngAGH5_4 and GH16 lichenases both hydrolyze MLG, these two types of enzymes release different series of oligosaccharides. Structural analyses of ZgEngAGH5_4 reveal that all the amino acid residues involved in the catalytic triad and in the negative glucose-binding subsites are conserved, when compared with the closest relative, the cellulase EngD from Clostridium cellulovorans, and some other GH5s. In contrast, the positive glucose-binding subsites of ZgEngAGH5_4 are different and this could explain the preference for MLG, with respect to cellulose or laminarin. Molecular dynamics computer simulations using different hexaoses reveal that the specificity for MLG occurs through the +1 and +2 subsites of the binding pocket that display the most important differences when compared with the structures of other GH5_4 enzymes.

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.

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 CglA, 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 a 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 CglA hydrolyses the beta-(1-->4) linkage of lambda-carrageenan. LC-MALLS (liquid chromatography-multi-angle laser light scattering) 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-lambda-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.