The role of amino acid residues in the active site of L-methionine γ-lyase from Pseudomonas putida.
Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine γ-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel β-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.
Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan. kinagaki@cc.okayama-u.ac.jp
Mitsuki
M
Fukumoto
Daizou
D
Kudou
Shouko
S
Murano
Tomoo
T
Shiba
Dan
D
Sato
Takashi
T
Tamura
Shigeharu
S
Harada
Kenji
K
Inagaki
D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013437:Carbon-Sulfur Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016958:Pseudomonas putida / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D001224:Aspartic Acid; D006639:Histidine; D008715:Methionine; D013437:Carbon-Sulfur Lyases; C021558:L-methionine gamma-lyase; D008239:Lysine; D003545:Cysteine