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J. Biol. Chem., Vol. 282, Issue 16, 12220-12229, April 20, 2007

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Crystal Structure of Helicobacter pylori Formamidase AmiF Reveals a Cysteine-Glutamate-Lysine Catalytic Triad^*

Chiu-Lien Hung, Jia-Hsin Liu, Wei-Chun Chiu, Shao-Wei Huang, Jenn-Kang Hwang¹, and Wen-Ching Wang²

From the Institute of Molecular and Cellular Biology and Department of Life Science, National Tsing Hua University, Hsinchu 300, Taiwan and the Institute of Bioinformatics, National Chiao Tung University, Hsinchu 300, Taiwan

Helicobacter pylori AmiF formamidase that hydrolyzes formamide to produce formic acid and ammonia belongs to a member of the nitrilase superfamily. The crystal structure of AmiF was solved to 1.75Å resolution using single-wavelength anomalous dispersion methods. The structure consists of a homohexamer related by 3-fold symmetry in which each subunit has an --- four-layer architecture characteristic of the nitrilase superfamily. One exterior layer faces the solvent, whereas the other one associates with that of the neighbor subunit, forming a tight ------- dimer. The apo and liganded crystal structures of an inactive mutant C166S were also determined to 2.50 and 2.30Å_, respectively. These structures reveal a small formamide-binding pocket that includes Cys¹⁶⁶, Glu⁶⁰, and Lys¹³³ catalytic residues, in which Cys¹⁶⁶ acts as a nucleophile. Analysis of the liganded AmiF and N-carbamoyl D-amino acid amidohydrolase binding pockets reveals a common Cys-Glu-Lys triad, another conserved glutamate, and different subsets of ligand-binding residues. Molecular dynamic simulations show that the conserved triad has minimal fluctuations, catalyzing the hydrolysis of a specific nitrile or amide in the nitrilase superfamily efficiently.

Received for publication, September 26, 2006 , and in revised form, January 18, 2007.

The atomic coordinates and structure factors (code 2DYU, 2E2K, and 2E2L) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Science Council, Taiwan, Grants NSC95-2313-B-007-001, NSC94-2313-B-007-002, NSC94-3112-B-007-005, and NSC95-3112-B-007-002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed. E-mail: jkhwang{at}faculty.nctu.edu.tw.

² To whom correspondence may be addressed. E-mail: wcwang{at}life.nthu.edu.tw.

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