Increasing the flexibility of the substrate binding pocket of Streptomyces phospholipase D can enhance its catalytic efficiency in soybean phosphatidylcholine.

Autor: Hu R; Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Metabolic Diseases, College of Life Sciences, Anhui Normal University, Wuhu 241000, Anhui, PR China; Auhui Provincial Engineering Research Centre for Molecular Detection and Diagnostics, College of Life Sciences, Anhui Normal University, Wuhu 241000, Anhui, PR China. Electronic address: Rongkang_Hu@163.com., Cao J; Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Metabolic Diseases, College of Life Sciences, Anhui Normal University, Wuhu 241000, Anhui, PR China; Auhui Provincial Engineering Research Centre for Molecular Detection and Diagnostics, College of Life Sciences, Anhui Normal University, Wuhu 241000, Anhui, PR China., Rong C; Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Metabolic Diseases, College of Life Sciences, Anhui Normal University, Wuhu 241000, Anhui, PR China; Auhui Provincial Engineering Research Centre for Molecular Detection and Diagnostics, College of Life Sciences, Anhui Normal University, Wuhu 241000, Anhui, PR China., Wu S; Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Metabolic Diseases, College of Life Sciences, Anhui Normal University, Wuhu 241000, Anhui, PR China; Auhui Provincial Engineering Research Centre for Molecular Detection and Diagnostics, College of Life Sciences, Anhui Normal University, Wuhu 241000, Anhui, PR China., Wu L; Translational Medicine Center, The Second Affiliated Hospital of Wannan Medical College, Wuhu, Anhui 241000, PR China. Electronic address: linxiu0508@wnmc.edu.cn.
Jazyk: angličtina
Zdroj: International journal of biological macromolecules [Int J Biol Macromol] 2024 Nov; Vol. 280 (Pt 2), pp. 135824. Date of Electronic Publication: 2024 Sep 19.
DOI: 10.1016/j.ijbiomac.2024.135824
Abstrakt: 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.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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