title: The role of active site tyrosine 58 in Citrobacter freundii methionine γ-lyase.
abstract: In the spatial structure of methionine γ-lyase (MGL, EC 4.4.1.11) from Citrobacter freundii, Tyr58 is located at H-bonding distance to the oxygen atom of the phosphate "handle" of pyridoxal 5'-phosphate (PLP). It was replaced for phenylalanine by site-directed mutagenesis. The X-ray structure of the mutant enzyme was determined at 1.96Å resolution. Comparison of spatial structures and absorption spectra of wild-type and mutant holoenzymes demonstrated that the replacement did not result in essential changes of the conformation of the active site Tyr58Phe MGL. The Kd value of PLP for Tyr58Phe MGL proved to be comparable to the Kd value for the wild-type enzyme. The replacement led to a decrease of catalytic efficiencies in both γ- and β-elimination reactions of about two orders of magnitude as compared to those for the wild-type enzyme. The rates of exchange of C-α- and C-β- protons of inhibitors in D2O catalyzed by the mutant form are comparable with those for the wild-type enzyme. Spectral data on the complexes of the mutant form with the substrates and inhibitors showed that the replacement led to a change of rate the limiting step of the physiological reaction. The results allowed us to conclude that Tyr58 is involved in an optimal positioning of the active site Lys210 at some stages of γ- and β-elimination reactions. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow 119991, Russia.
authors.forename: Natalya V
authors.initials: NV
authors.lastname: Anufrieva
authors.affiliation: A.N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, ul. Vavilova 28, Moscow 117813, Russia.
authors.forename: Nicolai G
authors.initials: NG
authors.lastname: Faleev
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow 119991, Russia.
authors.forename: Elena A
authors.initials: EA
authors.lastname: Morozova
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow 119991, Russia.
authors.forename: Natalia P
authors.initials: NP
authors.lastname: Bazhulina
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow 119991, Russia.
authors.forename: Svetlana V
authors.initials: SV
authors.lastname: Revtovich
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow 119991, Russia.
authors.forename: Vladimir P
authors.initials: VP
authors.lastname: Timofeev
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow 119991, Russia.
authors.forename: Yaroslav V
authors.initials: YV
authors.lastname: Tkachev
authors.affiliation: Institute of Protein Research, Russian Academy of Sciences, ul. Institutskaya 4, Pushchino, Moscow Region 142290, Russia.
authors.forename: Alexei D
authors.initials: AD
authors.lastname: Nikulin
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow 119991, Russia. Electronic address: tvd@eimb.ru.
authors.forename: Tatyana V
authors.initials: TV
authors.lastname: Demidkina
mesh_terms: D013437:Carbon-Sulfur Lyases / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D016971:Citrobacter freundii / Q000201:enzymology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D014443:Tyrosine
chemical_list: D014443:Tyrosine; D013437:Carbon-Sulfur Lyases; C021558:L-methionine gamma-lyase
keywords: Cofactor-binding residue; Guiding role; Methionine γ-lyase; Mutant form; Pyridoxal 5′-phosphate; Three-dimensional structure