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Green algae belonging to the genus Ulvales are known to produce ulvan which is one of the main polysaccharide components of their cell wall. Ulvan is composed of 3O-sulfate-rhamnose (Rha3S), glucuronic acid (GlcA), iduronic acid (IduA) and xylose (Xyl) distributed in three disaccharide repetition moieties: [→ 4)- β -D-GlcA-(1 → 4)- α -L-Rha3S-(1 →], [→ 4)- α -L-IdoA-(1 → 4)- α -L-Rha3S(1 →] and [→ 4)- β -D-Xyl-(1 → 4)- α -L-Rha3S(1 →]. The ability of bacteria to degrade complex algal polysaccharides such as ulvan is usually encoded in clusters of genes referred to as polysaccharide utilization loci (PUL). Full saccharification of ulvan is expected to require an ulvan lyase, which cleaves the β -(1 → 4)-glycosidic bond between Rha3S and GluA or IduA through a β -elimination mechanism. In addition, enzymes with β -glucuronyl hydrolase, rhamnosidase, xylosidase and sulfatase activity are also expected. Recently, the genomes of several ulvan degrading bacteria were sequenced, which led to the identification of a new family of polysaccharide lyases family 24 (PL24). In this work, we have continued to mine the genomic data of one of the sequenced strains, Alteromonas sp. LOR. Here we report the identification of an ulvan associated PUL residing between open reading frames ( lor_19 – lor_34 ). This PUL contains a TonB dependent receptor, along with an experimentally verified rhamnosidase, a β -glucuronyl hydrolase and predicted sulfatases. Interestingly, we also identified in the PUL a new ulvan lyase (LOR_29) which showed no homology to previously reported ulvan lyases making it a founding member of yet another new family of polysaccharide lyases (PL25). Finally, this enzyme prompted us to mine other genomes where we identified additional potential ulvan PULs harboring this gene in other bacterial species. Taken together our report provides further insight into ulvan degradation mechanisms in bacteria and reveals a new family of polysaccharide lyases. more...

Ulvan is a complex sulfated polysaccharide biosynthesized by green seaweed and contains predominantly rhamnose, xylose, and uronic acid sugars. Ulvan-degrading enzymes have only recently been identified and added to the CAZy ( www.cazy.org ) database as family PL24, but neither their structure nor catalytic mechanism(s) are yet known. Several homologous, new ulvan lyases, have been discovered in Pseudoalteromonas sp. strain PLSV, Alteromonas LOR, and Nonlabens ulvanivorans, defining a new family PL25, with the lyase encoded by the gene PLSV_3936 being one of them. This enzyme cleaves the glycosidic bond between 3-sulfated rhamnose (R3S) and glucuronic acid (GlcA) or iduronic acid (IdoA) via a β-elimination mechanism. We report the crystal structure of PLSV_3936 and its complex with a tetrasaccharide substrate. PLSV_3936 folds into a seven-bladed β-propeller, with each blade consisting of four antiparallel β-strands. Sequence conservation analysis identified a highly conserved region lining at one end of a deep crevice on the protein surface. The putative active site was identified by mutagenesis and activity measurements. Crystal structure of the enzyme with a bound tetrasaccharide substrate confirmed the identity of base and acid residues and allowed determination of the catalytic mechanism and also the identification of residues neutralizing the uronic acid carboxylic group. The PLSV_3936 structure provides an example of a convergent evolution among polysaccharide lyases toward a common active site architecture embedded in distinct folds. more...