A product-determining structural loop in a novel PL40 ulvan lyase controls oligosaccharide profile.
TL;DR
It is shown that variations in the architecture of the substrate-binding cleft dictate whether these enzymes generate disaccharides or longer oligosaccharides as their dominant products, helping to understand how PL40 enzymes work and offering a starting point for designing ulvan lyases that make specific products.
Abstract
Ulvan lyases play a key role in marine polysaccharide degradation, but how members of the PL40 family achieve their specific product profiles remains poorly understood. Here, we show that variations in the architecture of the substrate-binding cleft dictate whether these enzymes generate disaccharides or longer oligosaccharides as their dominant products. We focused on JpPL40A, a previously uncharacterized ulvan lyase from the marine bacterium Jejuia pallidilutea, and found that it works best at 40 °C and pH 8.0. Using a combination of homology modeling and site-directed mutagenesis, the catalytic mechanism was elucidated, identifying a functional Tyr/His pair (Y260/H429) with R459 responsible for neutralizing the substrate's carboxyl group. Notably, comparative structural analysis revealed that a unique loop region adjacent to substrate binding subsites +5 and +6 dictates its product profile, leading to the predominant generation of unsaturated hexasaccharides. This loop's crucial role was confirmed via truncation mutagenesis, which shifted the main product to disaccharides, and further validated by the discovery of a homologous lyase sharing this structural feature. These results reveal a new structural feature that controls product specificity, helping us better understand how PL40 enzymes work and offering a starting point for designing ulvan lyases that make specific products.