Abstract
Alginate is a major component of brown algae cell walls and can be degraded via β-elimination by alginate lyases. These enzymes are classified into polysaccharide lyases and oligo-alginate lyases (Oals), with Oals mainly represented by the PL15 and PL17 families. Unlike PL17 Oals, which are widely present in alginate-degrading microorganisms, PL15 enzymes are only identified in a limited number of microorganisms, and their biochemical characteristics remain poorly understood. In this research, a novel PL15 alginate lyase, VBAly15A, from the marine bacterium, Vibrio sp. B1Z05, was identified and characterized. It belongs to a new PL15_3 subfamily and exhibits high activity toward polyM substrates. VBAly15A is thermostable in medium temperatures, tolerant to alkaline up to 11.0, and polyM-specific Oal, and it can first degrade alginate polymers into disaccharides and subsequently catalyze disaccharides into monomers via an exolytic mode. Site-directed mutagenesis showed that Arg(114), Tyr(470), and Arg(110) in the active groove are essential for the stable binding of the substrate. In addition, the amino acid His(226) in VBAly15A, previously suggested to act as a catalytic base, is not essential for catalysis, whereas Tyr(280), previously proposed to act as a catalytic acid, is required for enzyme activity. Structural bioinformatic and biochemical analyses revealed that His(226) functions as a catalytic base, specifically abstracting protons from G-type substrates, while Tyr(280) acts as both a catalytic acid and a base. This catalytic mechanism is likely conserved in PL15 family alginate lyases.IMPORTANCEAlginate, as a renewable resource for sustainability, has great application prospects. In addition to polysaccharide lyases, Oals are critical for the full degradation of alginate, a key prerequisite for biorefinery. So far, most identified and well-characterized Oals belong to the PL17 family. However, the catalytic mechanism of PL15 Oals is limited, and even the catalytic base and acid are not fully elucidated. The significance of this study lies in discovering and characterizing a novel Oal VBAly15A that divides into a new PL15 subfamily, PL15_3. Not only are key amino acid residues involved in enzyme activity identified, but residues acting as the catalytic base and acid are also demonstrated. The distance of the catalytic residues His and Tyr to the C5 proton of the sugar ring determines the substrate specificity. Therefore, this work provides new insights into the mechanism of substrate specificity in alginate lyases.