Substitution of a non-active-site residue located on the T3 loop increased the catalytic efficiency of endo -polygalacturonases

Endo-polygalacturonases (PGs) of glycosyl hydrolase (GH) family 28 share a right handed parallel β-helical structure with a cleft formed by T1 and T3 loops. To reveal the effect of non-active-site residues on endo-PG catalysis, Thr113 of Achaetomium sp. endo-PG (PG8fn) located on the T3 loop was substituted. The experimental results indicated that the catalytic efficiency of PG8fn depends on the side-chain structure of residue at position 113, following the order of Glu (negatively charged) < Gly, Ala and Ile (nonpolar) < Ser, Gln and Thr (polar) < Arg and Lys (positively charged). Void pathway analysis demonstrated that substitution with Arg caused the increased occupancy rates of void pathways T2 and T3 and resulted in the improvement of catalytic efficiency by increasing the kinetics of substrate access (kon) or product release (koff). Further molecular dynamics (MD) simulations illustrated that the substitution enhanced the flexibility of T3 loop and influenced the interaction between enzyme and substrate. The functional role of Arg113 was also verified in another GH28 endo-PG by increasing the catalytic efficiency of ∼2.4-fold. This study proposes a novel strategy, i.e. selective modification of the non-active site residues, to improve the catalytic performance of GH28 PGs.