Purification and biochemical characterization of the hydantoin hydrolyzing enzyme from Agrobacterium species

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Abstract
A soluble hydantoinase (5,6-dihydropyrimidine amidohydrolase) was purified to homogeneity from a newly isolated Agrobacterium species. This hydrolase consists of about 578 aminoacyl residues and is a slightly acidic protein with an isoelectric point of 6.5. The first 22 N-terminal amino acid residues were determined by Edman degradation. Determination of the relative molecular mass of the protein by gel-filtration chromatography gave an apparent value of 250000. The subunit Mr was 62000, as estimated by analytical SDS/PAGE and 66500, as estimated by denaturing gel-filtration chromatography. The pure hydantoinase exhibits the following hydrodynamic properties: a sedimentation coefficient of 8.8 S as determined by sedimentation velocity experiments; a Stokes radius of 6.8 nm; a diffusion coefficient of 31.5 μm2·s−1 as determined by analytical gelfiltration chromatography. From these experimental data, the following physical constants could be calculated: a theoretical Mr of 265000, a frictional ratio, f/fo, of 1.59, a maximal axial ratio, a/b, of 3.1; a Perrin shape factor, F, of 1.37. As shown by different Km values, the preferred substrates of this hydrolase were 5-monosubstituted hydantoins bearing aromatic substituents. 5,5-Dimethylhydantoin and different thio analogs of the 5-p-hydroxyphenylhydantoin molecule are competitive inhibitors of this hydrolase. The classification of this microbial hydantoinase, which exhibits no hydrolytic activity with all the dihydropyrimidines tested, under the systematic name of 5,6-dihydropyrimidine amidohydrolase, and its putative metabolic role are further discussed.