Increasing the flexibility of the substrate binding pocket of Streptomyces phospholipase D can enhance its catalytic efficiency in soybean phosphatidylcholine
Rongkang Hu, Jiale Cao, Chenghao Rong, Siyi Wu, Linxiu Wu
Abstract
The catalytic efficiency of Streptomyces klenkii phospholipase D (SkPLD) in soybean phosphatidylcholine (soy-PC) processing is constrained by its acyl chain specificity. To address this limitation, we engineered the substrate-binding pocket of SkPLD to increase its flexibility. The mutant P343A/Y383L exhibited a 7.14-fold increase in catalytic efficiency toward soy-PC compared to the wild type. This enhancement was attributed to improved substrate-binding pocket flexibility, as evidenced by the significantly higher specific activity of the mutant toward PCs with various acyl chains (58.20–327.76 U/mg vs. 13.56–76.67 U/mg). Monomolecular film experiments demonstrated that the P343A/Y383L mutant reduced the energy barrier for PC binding, facilitating favorable interactions with the soy-PC monolayer. Molecular dynamics simulations revealed that the mutant's increased flexibility allowed for easier diffusion and penetration into the soy-PC monolayer, while the non-polar amino acids in the substrate-binding pocket promoted rapid interactions with the acyl chains of PC, ultimately leading to enhanced catalytic activity. • The P343A/Y383L mutant's catalytic efficiency was 7.14 times that of the wild type. • The wild type (SkPLD) substrate selectivity was attenuated by two-point mutation. • The mutant P343A/Y383L improved SkPLD adsorption stability and binding affinity. • Phosphatidylcholines with short acyl chain were more chemically stable and reactive. • Improving hydrophobicity and available volume increased binding pocket's flexibility.