Citrobacter freundii Methionine γ-Lyase: The Role of Serine 339 in the Catalysis of γ- and β-Elimination Reactions.

Autor:	Anufrieva NV; Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences, Moscow, 119991 Russia., Morozova EA; Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences, Moscow, 119991 Russia., Revtovich SV; Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences, Moscow, 119991 Russia., Bazhulina NP; Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences, Moscow, 119991 Russia., Timofeev VP; Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences, Moscow, 119991 Russia., Tkachev YV; Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences, Moscow, 119991 Russia., Faleev NG; Nesmeyanov Institute of Organoelement Compounds of the Russian Academy of Sciences, Moscow, 119991 Russia., Nikulin AD; Institute of Protein Research of the Russian Academy of Sciences, Pushchino, Moscow Region, 142290 Russia., Demidkina TV; Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences, Moscow, 119991 Russia.
Jazyk:	angličtina
Zdroj:	Acta naturae [Acta Naturae] 2022 Apr-Jun; Vol. 14 (2), pp. 50-61.
DOI:	10.32607/actanaturae.11242
Abstrakt:	Serine 339 of the active site of Citrobacter freundii methionine γ-lyase (MGL) is a conserved amino acid in most pyridoxal 5'-phosphate-dependent enzymes of the cystathionine β-lyase subclass, to which MGL belongs. The reaction mechanism of the MGL-catalyzed γ-elimination reaction is poorly explored. We replaced serine 339 with alanine using site-directed mutagenesis. The replacement of serine 339 with alanine led to a significant (by two orders of magnitude) decrease in efficiency in the catalysis of the γ- and β-elimination reactions by the mutant form of the enzyme. The exchange rates of the C-α- and C-β-protons in the amino acids in complexes consisting of the enzyme and competitive inhibitors decreased by one-two orders of magnitude. The spectral characteristics of the mutant form indicated that the replacement did not lead to significant changes in the conformation and tautomerism of MGL internal aldimine. We crystallized the holoenzyme and determined its spatial structure at 1.7 E resolution. The replacement of serine 339 with alanine did not affect the overall course of the polypeptide chain of the MGL subunit and the tetrameric enzyme structure. An analysis of the obtained kinetic and spectral data, as well as the known spatial structures of C. freundii MGL, indicates that serine 339 is necessary for efficient catalysis of γ- and β-elimination reactions at the stage of C-α-proton abstraction from the external aldimine, the γ-elimination reaction at the stages of coenzyme C4'-atom protonation, and C-β-proton abstraction from a ketimine intermediate. (Copyright ® 2022 National Research University Higher School of Economics.)
Databáze:	MEDLINE
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