Structural and biochemical characterization of the laminarinaseZgLamC_GH16fromZobellia galactanivoranssuggests preferred recognition of branched laminarin

Abstract

Laminarin is a β-1,3-D-glucan displaying occasional β-1,6 branches. This storage polysaccharide of brown algae constitutes an abundant source of carbon for marine bacteria such as Zobellia galactanivorans. This marine member of the Bacteroidetes possesses five putative β-1,3-glucanases [four belonging to glycosyl hydrolase family 16 (GH16) and one to GH64] with various modular architectures. Here, the characterization of the β-glucanase ZgLamC is reported. The catalytic GH16 module (ZgLamCGH16) was produced in Escherichia coli and purified. This recombinant enzyme has a preferential specificity for laminarin but also a significant activity on mixed-linked glucan (MLG). The structure of an inactive mutant of ZgLamCGH16 in complex with a thio-β-1,3-hexaglucan substrate unravelled a straight active-site cleft with three additional pockets flanking subsites -1, -2 and -3. These lateral pockets are occupied by a glycerol, an acetate ion and a chloride ion, respectively. The presence of these molecules in the vicinity of the O6 hydroxyl group of each glucose moiety suggests that ZgLamCGH16 accommodates branched laminarins as substrates. Altogether, ZgLamC is a secreted laminarinase that is likely to be involved in the initial step of degradation of branched laminarin, while the previously characterized ZgLamA efficiently degrades unbranched laminarin and oligo-laminarins.

Molecular Mechanism by which Prominent Human Gut Bacteroidetes Utilize Mixed-Linkage Beta-Glucans, Major Health-Promoting Cereal Polysaccharides

“…The apo structure of recBoGH16 MLG was determined to a resolution of 1.8 Å by molecular replacement using the structure of Zobellia galactanivorans laminarinase ZgLamC GH16-E142S (PDB code 4CRQ) (Labourel et al, 2015) as a search model (See Table S4 for processing and refinement statistics). The crystal contained two polypeptide chains in the asymmetric unit corresponding to residues I35-L271 of wild-type BoGH16 MLG for both chains (residue numbering is from transcriptional start site according to the genomic sequence).…”

“…Specifically, the top match (Z-score = 29.3) was the structure of laminarinase “ZgLamC GH16-E142S “ from Zobellia galactanivorans (PDB code 4CTE) (Labourel et al, 2015), which has 38% amino acid identity and superimposed with BoGH16 MLG with a root mean square deviation (RMSD) value of 2.0 Å over 211 out of 231 Cα pairs. In comparison, the closest GH16 homolog with a regular active-site β-strand was the lichenase (MLGase) from Paenibacillus macerans (PDB code 1MAC) (Hahn et al, 1995), which has a comparable Z-score of 25.1 and an RMSD value of also 2.0 Å over 200 out of 212 Cα pairs, despite having only 22% amino acid identity with BoGH16 MLG .…”

“…Such curvature is exemplified by the ZgLamC GH16-E142S :thio-β-1,3-trisaccharide structure (Fig. S7A, PDB code 4CTE) (Labourel et al, 2015). Indeed, Ilari et al observed that shortening the homologous loop in LamA from the archeon Pyrococcus furiosus (Fig.…”

Alpha‐ and beta‐mannan utilization by marine Bacteroidetes

“…S6). These putative laminarin PULs encode glycoside hydrolases that have been previously shown to be involved in laminarin degradation by proteomics (Kabisch et al ., ; Xing et al ., ) and biochemical analyses (Labourel et al ., ; ; Unfried et al ., ). We therefore used laminarin degradation activity as a functionally characterized positive control.…”

The Mannitol Utilization System of the Marine Bacterium Zobellia galactanivorans

“…Indeed, it possesses two alginolytic operons induced by the presence of alginate (43), and the first two alginate lyases of this complex system (AlyA1 and AlyA5) have recently been characterized at the biochemical and structural levels (44). This bacterium can also grow with brown algal laminarin as the sole carbon source, and among the five putative laminarinases identified in its genome (GenBank accession number FP476056), two GH16 laminarinases, Z. galactanivorans LamA (ZgLamA) and ZgLamC, have recently been analyzed biochemically and structurally (45). In addition to alginate and laminarin, mannitol is one of the most abundant carbohydrates in brown algae, and the annotation of the Z. galactanivorans genome has suggested the presence of proteins potentially involved in the use of this storage compound.…”