Abstract
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 1.75A resolution using single-wavelength anomalous dispersion methods. The structure consists of a homohexamer related by 3-fold symmetry in which each subunit has an alpha-beta-beta-alpha four-layer architecture characteristic of the nitrilase superfamily. One exterior alpha layer faces the solvent, whereas the other one associates with that of the neighbor subunit, forming a tight alpha-beta-beta-alpha-alpha-beta-beta-alpha dimer. The apo and liganded crystal structures of an inactive mutant C166S were also determined to 2.50 and 2.30 A, respectively. These structures reveal a small formamide-binding pocket that includes Cys(166), Glu(60), and Lys(133) catalytic residues, in which Cys(166) 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.
Highlights
The first determined structure of the nitrilase (Nit) domain is from worm NitFhit that shows a tetramer, and each Nit domain has a four-layer ␣---␣ sandwich fold [3]
Structural and site-directed mutagenesis results suggest the roles of the CEK residues; the active cysteine acts as the nucleophile, glutamate mediates the proton transfer, and lysine stabilizes a tetrahedral transition state [6, 7]
AmiF and the other paralogue AmiE, along with urease, play a major role in producing ammonia to protect against gastric acidity or as a nitrogen source or a cytotoxic molecule, enabling H. pylori to adapt into such an exclusive environment [11, 12]
Summary
Site-directed Mutagenesis, Expression, and Purification—The gene (amiF) encoding formamidase was amplified from chromosomal DNA of H. pylori 26695 by PCR using pfu DNA polymerase and inserted into the pQE30 expression vector to generate pQE30-AmiF. The native crystal was characterized as belonging to space group R3, with the unit cell dimensions a ϭ b ϭ 147.21, c ϭ 72.27 Å. The thin plate crystal was characterized as space group C2, with the unit cell dimensions of a ϭ 117.72, b ϭ 130.53, c ϭ 144.59 Å, and  ϭ 99.44°. The C166S complex crystal was obtained by the co-crystallization method in a solution containing 0.1 M sodium acetate (pH 5.0), 0.2 M lithium sulfate, 6% PEG 1500, and 3 mM formamide. The complex crystal grew as a rod and was characterized as the space group P21 with cell dimensions of a ϭ 83.09, b ϭ 151.80, c ϭ 89.08 Å, and  ϭ 114.99°. The apo and liganded C166S structures were determined by the molecular
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