A key loop in the catalytic pocket of the PL17 family of alginate lyases determines minimal substrate recognition.

Alginate, a major component of brown macroalgae, is an alternative feedstock for biorefining. The degradation of alginate oligosaccharides (AOSs) is a key prerequisite for biorefining, which usually requires at least two oligo-alginate lyases (Oals). However, the function and minimal substrate recognition mechanisms of different Oals in alginate metabolism remain poorly understood. In this study, a pair of PL17 family Oals (VaAly17A and VaAly17B) was identified, which is universal in alginate-degrading Vibrio species. VaAly17A is crucial for alginate metabolism, primarily acting on substrates larger than disaccharides, while VaAly17B contributes to rapid alginate utilization by converting disaccharides into monomers. The distinct minimal degrading substrates of the two alginate lyases are determined by a critical loop, Loop1, around the active groove. VaAly17A, with a shorter Loop1, forms an open groove for binding larger substrates, while the longer Loop1 in VaAly17B results in a shorter catalytic cleft that accommodates only smaller substrates like disaccharides. Loop swapping experiments indicate that the shorter Loop1 is crucial for interacting with larger substrates, and structure alignment suggests that this loop may serve as a hallmark to distinguish the minimal substrates among PL17 Oals. Altogether, this study, for the first time, identifies a loop of PL17 Oals determining minimal substrate recognition and provides a new strategy for distinguishing the minimal recognition patterns of PL17 Oals.