The role of amino acid residues in the active site of L-methionine γ-lyase from Pseudomonas putida

Mitsuki F. Ukumoto, Daizou K. Udou, Shouko M. Urano, Tomoo S. Hiba, Dan Sato, Takashi Tamura, Shigeharu Harada, Kenji Inagaki

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1275-1284
Number of pages10
JournalBioscience, Biotechnology and Biochemistry
Volume76
Issue number7
DOIs
Publication statusPublished - Jul 30 2012

Keywords

  • Crystal structure
  • L-methionine γ-lyase
  • Mutagenesis
  • Reaction specificity

ASJC Scopus subject areas

  • Biotechnology
  • Analytical Chemistry
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Organic Chemistry

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