Dissecting a two-domain alginate lyase of family PL6 reveals a mechanistic basis for substrate specificity and enzyme activity

Alginate lyases (ALs) cleave 4-O-glycosidic linkages in alginate, composed of mannuronate (M) and guluronate (G) residues via β-elimination with preference for either one or several M-M, M-G, G-M, G-G linkages. ALs in polysaccharide lyase family 6 (PL6) present different specificities and modes of action and contain either one or two parallel β-helix domains. About half of almost 600 PL6 sequences are of the two-domain type, all located in the phyla Pseudomonadota and Bacteroidota. Here, functional roles are described for the N- and C-terminal domains (NTD and CTD) using BoPL6, a two-domain AL from the human gut bacterium Bacteroides ovatus CP926, which is specific for G in subsite +1. The NTD contains the catalytic site, but BoPL6-NTD markedly preferred the model substrate polyMG and cleaved M-G bonds in endo-mode, whereas the NTD + CTD mixture, similarly to BoPL6, acted with highest activity on model substrate polyG in exo-mode, verified by time-resolved ¹H-NMR. The CTD was not catalytically active but bound polyguluronate and, when mixed with BoPL6-NTD, promoted activity on polyG, yielding products of DP 1‒3, similarly to BoPL6. This defines a crucial role of the CTD in shaping the active site in BoPL6, as validated by substrate docking. The BoPL6 E634A mutant in the conserved CTD DEST loop, interacting with the active site in two-domain PL6 enzymes, was inactive, while the corresponding CTD mutant mixed with the NTD did not form the WT structure and had highly reduced activity on polyG but acted on polyMG in endo-mode with improved rate and conversion.