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Kinetic and structural studies on roles of the serine ligand and a strictly conserved tyrosine residue in nitrile hydratase

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Abstract

Nitrile hydratases (NHase), which catalyze the hydration of nitriles to amides, have an unusual Fe3+ or Co3+ center with two modified Cys ligands: cysteine sulfininate (Cys-SO2 −) and either cysteine sulfenic acid or cysteine sulfenate [Cys-SO(H)]. Two catalytic mechanisms have been proposed. One is that the sulfenyl oxygen activates a water molecule, enabling nucleophilic attack on the nitrile carbon. The other is that the Ser ligand ionizes the strictly conserved Tyr, activating a water molecule. Here, we characterized mutants of Fe-type NHase from Rhodococcus erythropolis N771, replacing the Ser and Tyr residues, αS113A and βY72F. The αS113A mutation partially affected catalytic activity and did not change the pH profiles of the kinetic parameters. UV–vis absorption spectra indicated that the electronic state of the Fe center was altered by the αS113A mutation, but the changes could be prevented by a competitive inhibitor, n-butyric acid. The overall structure of the αS113A mutant was similar to that of the wild type, but significant changes were observed around the catalytic cavity. Like the UV–vis spectra, the changes were compensated by the substrate or product. The Ser ligand is important for the structure around the catalytic cavity, but is not essential for catalysis. The βY72F mutant exhibited no activity. The structure of the βY72F mutant was highly conserved but was found to be the inactivated state, with αCys114-SO(H) oxidized to Cys-SO2 −, suggesting that βTyr72 affected the electronic state of the Fe center. The catalytic mechanism is discussed on the basis of the results obtained.

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Abbreviations

CtNHase:

Nitrile hydratase from Comamonas testosteroni Ni1

Cys-SO2H:

Cysteine sulfinic acid

Cys-SOH:

Cysteine sulfenic acid

HPLC:

High-performance liquid chromatography

n-BA:

n-Butyric acid

NHase:

Nitrile hydratase

PDB:

Protein Data Bank

PtNHase:

Nitrile hydratase from Pesudonocardia thermophila JCM 3095

ReNHase:

Nitrile hydratase from Rhodococcus erythropolis N771

RMSD:

Root-mean-square deviation

SCNase:

Thiocyanate hydrolase

t-BuCN:

tert-Butylnitrile

t-BuCONH2:

Trimethylacetamide

t-BuNC:

tert-Butylisonitrile

Tris:

Tris(hydroxymethyl)aminomethane

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Acknowledgments

We thank the beamline assistants at the Photon Factory for data collection at beamlines NW12A and BL5A. This work was supported in part by a Grant-in-Aid for Scientific Research from the Scientific Research (B) KAKENHI 19350080 (to M.O.) and (B) KAKENHI 21350089 (to M.O.). This work was performed with the approval of the Photon Factory Advisory Committee (approval no. 2008G640).

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Authors and Affiliations

  1. Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan

    Yasuaki Yamanaka, Koichi Hashimoto, Akashi Ohtaki, Keiichi Noguchi, Masafumi Yohda & Masafumi Odaka

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  1. Yasuaki Yamanaka
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  2. Koichi Hashimoto
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  3. Akashi Ohtaki
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  4. Keiichi Noguchi
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  5. Masafumi Yohda
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  6. Masafumi Odaka
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Correspondence to Masafumi Odaka.

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Yamanaka, Y., Hashimoto, K., Ohtaki, A. et al. Kinetic and structural studies on roles of the serine ligand and a strictly conserved tyrosine residue in nitrile hydratase. J Biol Inorg Chem 15, 655–665 (2010). https://doi.org/10.1007/s00775-010-0632-3

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