Exploring molecular determinants of polysaccharide lyase family 6–1 enzyme activity

Abstract

The Polysaccharide Lyase Family 6 (PL6) represents one of the 41 polysaccharide lyase families classified in the CAZy database with the vast majority of its members being alginate lyases grouped into three subfamilies, PL6_1–3. To decipher the mode of recognition and action of the enzymes belonging to subfamily PL6_1, we solved the crystal structures of Pedsa0632, Patl3640, Pedsa3628 and Pedsa3807, which all show different substrate specificities and mode of action (endo−/exo-lyase). Thorough exploration of the structures of Pedsa0632 and Patl3640 in complex with their substrates as well as docking experiments confirm that the conserved residues in subsites −1 to +3 of the catalytic site form a common platform which can accommodate various types of alginate in a very similar manner but with a series of original adaptations bringing them their specificities of action. From comparative studies with existing structures of PL6_1 alginate lyases, we observe that in the right-handed parallel β-helix fold shared by all these enzymes, the substrate binding site harbors the same overall conserved structures and organization. Despite this apparent similarity, it appears that members of the PL6_1 subfamily specifically accommodate and catalyze the degradation of different alginates suggesting that this common platform is actually a highly adaptable and specific tool.

Characterization of degradation patterns and enzymatic properties of a novel alkali-resistant alginate lyase AlyRm1 from Rubrivirga marina

“…The K m , V max and K cat values for sodium alginate were 12.6 mg/mL, 10.08 U/mg and 10.38 s −1 , respectively. As shown in Table 1 , most PL6 alginate lyases were specific for PolyMG or PolyG, such as OalS6, AlyMG and OalC6, certain enzymes, such as AlyF, AlyGC, Patl3640, and Pedsa0640 ( Violot et al, 2021 ) had no activity towards PolyM. In addition, Bcel PL6 is a PolyM-specific lyase with negligible PolyMG activity and no PolyG activity ( Stender et al, 2019 ).…”

Structural analyses of ‘substrate-pH of activity’ pairing observed in Polysaccharide lyases

“…The change in carboxylic group contribution can be attributed to change in ManA’s substrate pose in contrast to its C5-epimer GulA (Table S7, FIGURE 7E)) . Recently, the PLs from this family (PL-6) has also been implicated in cleavage of ManA containing substrate [30] . However, the catalytic Lys is in opposite direction of ManA’s C-5 orientation at [+1] subsite suggesting a highly dynamic binding site like their PL-5 counterpart.…”

“…The change in carboxylic group contribution can be attributed to change in ManA's substrate pose in contrast to its C5-epimer GulA (Table S7, FIGURE 7E)). Recently, the PLs from this family (PL-6) has also been implicated in cleavage of ManA containing substrate [30]. However, the subsite.…”

“…[30]. However, the Polysaccharide lyase's substrate-pH of activity pairing 20 catalytic Lys is in opposite direction of ManA's C-5 orientation at [+1] subsite suggesting a highly dynamic binding site like their PL-5 counterpart.PLs binding GulA at [+1] subsite displaying β-sandwich fold:The PL-7 family of PLs are known to specifically bind and cleave poly-GulA.…”

Structural Analyses of Substrate–pH Activity Pairing Observed across Diverse Polysaccharide Lyases

“…The lack of carboxylic group contribution can be attributed to ManA’s substrate pose in contrast to its C5-epimer GulA (Table S9, Figure E). Recently, the PLs from this family (PL-6) have also been implicated in the cleavage of ManA containing substrate . However, the catalytic Lys is in the opposite direction of ManA’s C-5 orientation at the [+1] subsite, suggesting a highly dynamic binding site like PL-5.…”