8JM5

Endo-deglycosylated hydroxynitrile lyase isozyme 5 mutant L331A/S333V/P340L from Prunus communis

Summary for 8JM5

• Entry DOI: 10.2210/pdb8jm5/pdb
• Descriptor: (R)-mandelonitrile lyase, FLAVIN-ADENINE DINUCLEOTIDE, DI(HYDROXYETHYL)ETHER, ... (6 entities in total)
• Functional Keywords: hydroxynitrile lyase, fad, hydrocynanation, lyase
• Biological source: Prunus dulcis (almond)
• Total number of polymer chains: 1
• Total formula weight: 61281.44

Authors

Zheng, Y.C.,Li, F.L.,Yu, H.L.,Xu, J.H. (deposition date: 2023-06-04, release date: 2024-06-05, Last modification date: 2026-01-07)

Primary citation

Zheng, Y.C.,Mao, Y.,Geng, Q.,Li, F.L.,Kong, X.D.,Qi, Y.K.,Zhang, L.,Chen, Q.,Zhang, Z.J.,Hong, R.,Zhao, Y.L.,Yu, H.L.,Xu, J.H.
Flipping of a Non-productive Substrate Binding Conformation Facilitates Hydroxynitrile Lyase Catalyzed Hydrocyanation.
Angew.Chem.Int.Ed.Engl., 64:e202515778-e202515778, 2025

PubMed Abstract: Understanding enzyme-substrate conformational transformations is crucial to the design and engineering of biocatalysts. However, the mechanisms by which substrates undergo dynamic transformations that regulate the function of an enzyme remain poorly understood. Hydroxynitrile lyase from Prunus communis (PcHNL5) catalyzes the cleavage of cyanohydrins. Its reverse reaction holds significant synthetic potential for the preparation of pharmaceutical precursors. Using a combination of crystallography and computational experiments, a novel flipped substrate binding state is identified within the substrate tunnel of the PcHNL5 mutant. This binding state is non-productive and undergoes a conformational change before the catalytic cycle can proceed. Site-saturation mutagenesis led to the discovery of a triple mutant, PcHNL5, that destabilizes the non-productive substrate binding state thereby facilitating its transition to the catalytically productive conformation and significantly enhancing catalytic efficiency. Crystallographic studies provide a structural description of the factors that stabilize versus destabilize the different binding conformers in the different enzyme variants and thus the differing catalytic efficiencies. These findings demonstrate that destabilizing unfavorable substrate binding conformations within an enzyme active site can improve functionality and provide a promising strategy for designing efficient biocatalysts.

PubMed: 41137404
DOI: 10.1002/anie.202515778

Experimental method

X-RAY DIFFRACTION (1.9 Å)