PLP-Dependent Enzyme Methionine γ-Lyase: Insights into the Michaelis Complex from Molecular Dynamics and Free Energy Simulations - Département de biologie
Journal Articles Biochemistry Year : 2023

PLP-Dependent Enzyme Methionine γ-Lyase: Insights into the Michaelis Complex from Molecular Dynamics and Free Energy Simulations

Abstract

Methionine γ-lyase (MGL) breaks down methionine, with the help of its cofactor pyridoxal-5′-phosphate (PLP), or vitamin B6. Methionine depletion is damaging for cancer cells but not normal cells, so MGL is of interest as a therapeutic protein. To increase our understanding and help engineer improved activity, we focused on the reactive, Michaelis complex between MGL, covalently bound PLP, and substrate Met. is not amenable to crystallography, as it proceeds to products. Experimental activity measurements helped exclude a mechanism that would bypass. We then used molecular dynamics and alchemical free energy simulations to elucidate its structure and dynamics. We showed that the PLP phosphate has a pK a strongly downshifted by the protein, whether Met is present or not. Met binding affects the structure surrounding the reactive atoms. With Met, the Schiff base linkage between PLP and a nearby lysine shifts from a zwitterionic, keto form to a neutral, enol form that makes it easier for Met to approach its labile, target atom. The Met ligand also stabilizes the correct orientation of the Schiff base, more strongly than in simulations without Met, and in agreement with structures in the Protein Data Bank, where the Schiff base orientation correlates with the presence or absence of a co-bound anion or substrate analogue in the active site. Overall, the Met ligand helps organize the active site for the enzyme reaction by reducing fluctuations and shifting protonation states and conformational populations.
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Dates and versions

hal-04659173 , version 1 (22-07-2024)

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Xingyu Chen, Nathan Ferchaud, Pierre Briozzo, David Machover, Thomas Simonson. PLP-Dependent Enzyme Methionine γ-Lyase: Insights into the Michaelis Complex from Molecular Dynamics and Free Energy Simulations. Biochemistry, 2023, 62 (18), pp.2791-2801. ⟨10.1021/acs.biochem.3c00355⟩. ⟨hal-04659173⟩
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