Dataset Information

Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase from Pseudomonas sp. strain ADP.

ABSTRACT: The allophanate hydrolase from Pseudomonas sp. strain ADP was expressed and purified, and a tryptic digest fragment was subsequently identified, expressed and purified. This 50?kDa construct retained amidase activity and was crystallized. The crystals diffracted to 2.5?Å resolution and adopted space group P21, with unit-cell parameters a = 82.4, b = 179.2, c = 112.6?Å, ? = 106.6°.

SUBMITTER: Balotra S

PROVIDER: S-EPMC3944691 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase from Pseudomonas sp. strain ADP.

Balotra Sahil S Newman Janet J French Nigel G NG Briggs Lyndall J LJ Peat Thomas S TS Scott Colin C

Acta crystallographica. Section F, Structural biology communications 20140219 Pt 3

The allophanate hydrolase from Pseudomonas sp. strain ADP was expressed and purified, and a tryptic digest fragment was subsequently identified, expressed and purified. This 50 kDa construct retained amidase activity and was crystallized. The crystals diffracted to 2.5 Å resolution and adopted space group P21, with unit-cell parameters a = 82.4, b = 179.2, c = 112.6 Å, β = 106.6°. ...[more]

PMID: 24598916

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Project description:AtzF, allophanate hydrolase, is a recently discovered member of the amidase signature family that catalyzes the terminal reaction during metabolism of s-triazine ring compounds by bacteria. In the present study, the atzF gene from Pseudomonas sp. strain ADP was cloned and expressed as a His-tagged protein, and the protein was purified and characterized. AtzF had a deduced subunit molecular mass of 66,223, based on the gene sequence, and an estimated holoenzyme molecular mass of 260,000. The active protein did not contain detectable metals or organic cofactors. Purified AtzF hydrolyzed allophanate with a k(cat)/K(m) of 1.1 x 10(4) s(-1) M(-1), and 2 mol of ammonia was released per mol allophanate. The substrate range of AtzF was very narrow. Urea, biuret, hydroxyurea, methylcarbamate, and other structurally analogous compounds were not substrates for AtzF. Only malonamate, which strongly inhibited allophanate hydrolysis, was an alternative substrate, with a greatly reduced k(cat)/K(m) of 21 s(-1) M(-1). Data suggested that the AtzF catalytic cycle proceeds through a covalent substrate-enzyme intermediate. AtzF reacts with malonamate and hydroxylamine to generate malonohydroxamate, potentially derived from hydroxylamine capture of an enzyme-tethered acyl group. Three putative catalytically important residues, one lysine and two serines, were altered by site-directed mutagenesis, each with complete loss of enzyme activity. The identity of a putative serine nucleophile was probed using phenyl phosphorodiamidate that was shown to be a time-dependent inhibitor of AtzF. Inhibition was due to phosphoroamidation of Ser189 as shown by liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry. The modified residue corresponds in sequence alignments to the nucleophilic serine previously identified in other members of the amidase signature family. Thus, AtzF affects the cleavage of three carbon-to-nitrogen bonds via a mechanism similar to that of enzymes catalyzing single-amide-bond cleavage reactions. AtzF orthologs appear to be widespread among bacteria.

Dataset Information

Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase from Pseudomonas sp. strain ADP.

Publications

Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase from Pseudomonas sp. strain ADP.

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