Structural and mechanistic insights into homocysteine degradation by a mutant of methionine γ-lyase based on substrate-assisted catalysis

Dan Sato, Tomoo Shiba, Shunsuke Yunoto, Kazuo Furutani, Mitsuki Fukumoto, Daizou Kudou, Takashi Tamura, Kenji Inagaki, Shigeharu Harada

Research output: Contribution to journalArticle

Abstract

Methionine γ-lyse (MGL) catalyzes the α, γ-elimination of l-methionine and its derivatives as well as the α, β-elimination of l-cysteine and its derivatives to produce α-keto acids, volatile thiols, and ammonia. The reaction mechanism of MGL has been characterized by enzymological studies using several site-directed mutants. The Pseudomonas putida MGL C116H mutant showed drastically reduced degradation activity toward methionine while retaining activity toward homocysteine. To understand the underlying mechanism and to discern the subtle differences between these substrates, we analyzed the crystal structures of the reaction intermediates. The complex formed between the C116H mutant and methionine demonstrated that a loop structure (Ala51–Asn64) in the adjacent subunit of the catalytic dimer cannot approach the cofactor pyridoxal 5′-phosphate (PLP) because His116 disrupts the interaction of Asp241 with Lys240, and the liberated side chain of Lys240 causes steric hindrance with this loop. Conversely, in the complex formed between C116H mutant and homocysteine, the thiol moiety of the substrate conjugated with PLP offsets the imidazole ring of His116 via a water molecule, disrupting the interaction of His116 and Asp241 and restoring the interaction of Asp241 with Lys240. These structural data suggest that the Cys116 to His mutation renders the enzyme inactive toward the original substrate, but activity is restored when the substrate is homocysteine due to substrate-assisted catalysis.

Original languageEnglish
Pages (from-to)1224-1230
Number of pages7
JournalProtein Science
Volume26
Issue number6
DOIs
Publication statusPublished - Jun 1 2017

Fingerprint

Lyases
Homocysteine
Catalysis
Methionine
Degradation
Pyridoxal Phosphate
Substrates
Sulfhydryl Compounds
Keto Acids
Pseudomonas putida
Derivatives
Reaction intermediates
Ammonia
Cysteine
Catalytic Domain
Dimers
Mutation
Crystal structure
Water
Enzymes

Keywords

  • pyridoxal 5′-phosphate
  • site-directed mutagenesis
  • substrate-assisted catalysis
  • sulfur-containing amino acids
  • X-ray crystal structure analysis

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Structural and mechanistic insights into homocysteine degradation by a mutant of methionine γ-lyase based on substrate-assisted catalysis. / Sato, Dan; Shiba, Tomoo; Yunoto, Shunsuke; Furutani, Kazuo; Fukumoto, Mitsuki; Kudou, Daizou; Tamura, Takashi; Inagaki, Kenji; Harada, Shigeharu.

In: Protein Science, Vol. 26, No. 6, 01.06.2017, p. 1224-1230.

Research output: Contribution to journalArticle

Sato, Dan ; Shiba, Tomoo ; Yunoto, Shunsuke ; Furutani, Kazuo ; Fukumoto, Mitsuki ; Kudou, Daizou ; Tamura, Takashi ; Inagaki, Kenji ; Harada, Shigeharu. / Structural and mechanistic insights into homocysteine degradation by a mutant of methionine γ-lyase based on substrate-assisted catalysis. In: Protein Science. 2017 ; Vol. 26, No. 6. pp. 1224-1230.
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