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Multi-method analysis revealed the mechanism of substrate selectivity in NHase: A gatekeeper residue at the activity center.

Authors :
Meng Y
Peplowski L
Wu T
Cheng Z
Han L
Qiao J
Cheng Z
Zhou Z
Source :
International journal of biological macromolecules [Int J Biol Macromol] 2024 Nov; Vol. 279 (Pt 3), pp. 135426. Date of Electronic Publication: 2024 Sep 07.
Publication Year :
2024

Abstract

Recognizing the critical need to elucidate the molecular determinants of this selectivity offers a pathway to engineer enzymes with broader and more versatile catalytic capabilities. Through integrated methods including phylogenetic analysis, molecular docking, and structural analysis, we identified a pivotal amino acid residue, αTrp116, linking the substrate binding pocket and the active site of a NHase from Pseudonocardia thermophila JCM 3095 (PtNHase). This residue acts as a crucial determinant of substrate specificity within the NHase enzyme. The mutant αW116R modified the substrate specificity of PtNHase, significantly enhancing its catalytic efficiency towards aromatic substrates. The catalytic activity for aromatic compounds such as 3-Cyanopyridine was 14-fold that of the wild-type, whereas its activity for aliphatic substrates diminished to one-sixth. MD simulations revealed that replacing αTrp116 with Arg allowed aromatic nitrile substrates to achieve more favorable conformations within the active site. Based on the mutant αW116R, we further constructed a combinatorial variant Pt-4, tailored for aromatic substrates, which exhibited an enzyme activity 50 times that of the wild-type. These results highlight the critical influence of amino acid residues in the enzyme's active site on substrate specificity and offer fresh perspectives and approaches for the evolution of enzymes.<br />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.<br /> (Copyright © 2024 Elsevier B.V. All rights reserved.)

Details

Language :
English
ISSN :
1879-0003
Volume :
279
Issue :
Pt 3
Database :
MEDLINE
Journal :
International journal of biological macromolecules
Publication Type :
Academic Journal
Accession number :
39251006
Full Text :
https://doi.org/10.1016/j.ijbiomac.2024.135426