Time-Resolved Crystallography of the Reaction Intermediate of Nitrile Hydratase: Revealing a Role for the Cysteinesulfenic Acid Ligand as a Catalytic Nucleophile.
The reaction mechanism of nitrile hydratase (NHase) was investigated using time-resolved crystallography of the mutant NHase, in which βArg56, strictly conserved and hydrogen bonded to the two post-translationally oxidized cysteine ligands, was replaced by lysine, and pivalonitrile was the substrate. The crystal structures of the reaction intermediates were determined at high resolution (1.2-1.3 Å). In combination with FTIR analyses of NHase following hydration in H2 (18) O, we propose that the metal-coordinated substrate is nucleophilically attacked by the O(SO(-) ) atom of αCys114-SO(-) , followed by nucleophilic attack of the S(SO(-) ) atom by a βArg56-activated water molecule to release the product amide and regenerate αCys114-SO(-) .
Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 (Japan).
Yasuaki
Y
Yamanaka
Division of Material Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602 (Japan).
Yuki
Y
Kato
Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 (Japan).
Koichi
K
Hashimoto
Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 (Japan).
Keisuke
K
Iida
Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 (Japan).
Kazuo
K
Nagasawa
Global Research Cluster Collaboration Promotion Unit, RIKEN, 2-1, Hirosawa, Wako-shi, Saitama 351-0198 (Japan).
Hiroshi
H
Nakayama
Global Research Cluster Collaboration Promotion Unit, RIKEN, 2-1, Hirosawa, Wako-shi, Saitama 351-0198 (Japan).
Naoshi
N
Dohmae
Instrumentation Analysis Center, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 (Japan).
Keiichi
K
Noguchi
Division of Material Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602 (Japan).
Takumi
T
Noguchi
Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 (Japan).
Masafumi
M
Yohda
Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588 (Japan). modaka@gipc.akita-u.ac.jp.|Department of Life Science, Faculty and Graduate School of Engineering and Resource Science, Akita University, Akita City, Akita 010-8502 (Japan). modaka@gipc.akita-u.ac.jp.
Masafumi
M
Odaka
D002384:Catalysis; D018360:Crystallography, X-Ray / Q000379:methods*; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry; D006836:Hydro-Lyases / Q000737:chemistry*; D008024:Ligands; D008958:Models, Molecular; D011487:Protein Conformation; D013434:Sulfenic Acids / Q000737:chemistry*
D008024:Ligands; D013434:Sulfenic Acids; D006836:Hydro-Lyases; C048831:nitrile hydratase; C100870:cysteinesulfenic acid; D003545:Cysteine
cysteinesulfenic acid; nitrile hydratase; reaction intermediates; reaction mechanisms; time-resolved X-ray crystallography