Molecular Insight into the Role of the N-terminal Extension in the Maturation, Substrate Recognition, and Catalysis of a Bacterial Alginate Lyase from Polysaccharide Lyase Family 18

Background: The maturation and catalysis mechanisms of the PL18 alginate lyases have not yet been reported. Results: The N-terminal extension in the precursor of PL18, aly-SJ02, helped the catalytic domain fold correctly. Key residues for substrate recognition and catalysis were determined. Conclusion: The catalytic mechanism of aly-SJ02 is proposed. Significance: This study provides the foremost insight into maturation and catalysis of PL18 alginate lyases. Bacterial alginate lyases, which are members of several polysaccharide lyase (PL) families, have important biological roles and biotechnological applications. The mechanisms for maturation, substrate recognition, and catalysis of PL18 alginate lyases are still largely unknown. A PL18 alginate lyase, aly-SJ02, from Pseudoalteromonas sp. 0524 displays a -jelly roll scaffold. Structural and biochemical analyses indicated that the N-terminal extension in the aly-SJ02 precursor may act as an intramolecular chaperone to mediate the correct folding of the catalytic domain. Molecular dynamics simulations and mutational assays suggested that the lid loops over the aly-SJ02 active center serve as a gate for substrate entry. Molecular docking and site-directed mutations revealed that certain conserved residues at the active center, especially those at subsites +1 and +2, are crucial for substrate recognition. Tyr(353) may function as both a catalytic base and acid. Based on our results, a model for the catalysis of aly-SJ02 in alginate depolymerization is proposed. Moreover, although bacterial alginate lyases from families PL5, 7, 15, and 18 adopt distinct scaffolds, they share the same conformation of catalytic residues, reflecting their convergent evolution. Our results provide the foremost insight into the mechanisms of maturation, substrate recognition, and catalysis of a PL18 alginate lyase.