PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF THE HYDANTOIN HYDROLYZING ENZYME FROM AGROBACTERIUM SPECIES - A HYDANTOINASE WITH NO 5,6-DIHYDROPYRIMIDINE AMIDOHYDROLASE ACTIVITY

A soluble hydantoinase (5,6-dihydropyrimidine amidohydrolase) was puri fied to homogeneity from a newly isolated Agrobacterium species. This hydrolase consists of about 578 aminoacyl residues and is a slightly a cidic protein with an isoelectric point of 6.5. The first 22 N-termina l amino acid residues were determined by Edman degradation. Determinat ion of the relative molecular mass of the protein by gel-filtration ch romatography gave an apparent value of 250000. The subunit M(r) was 62 000, as estimated by analytical SDS/PAGE and 66500, as estimated by de naturing 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 r adius of 6.8 nm; a diffusion coefficient of 31.5 mum2.s-1 as determine d by analytical gel-filtration chromatography. From these experimental data, the following physical constants could be calculated: a theoret ical M(r) of 265000, a frictional ratio, f/fo, of 1.59, a maximal axia l ratio, alb, of 3.1; a Perrin shape factor, F, of 1.37. As shown by d ifferent K(m) values, the preferred substrates of this hydrolase were 5-monosubstituted hydantoins bearing aromatic substituents. 5,5-Dimeth ylhydantoin and different thio analogs of the 5-p-hydroxyphenylhydanto in molecule are competitive inhibitors of this hydrolase. The classifi cation of this microbial hydantoinase, which exhibits no hydrolytic ac tivity with all the dihydropyrimidines tested, under the systematic na me of 5,6-dihydropyrimidine amidohydrolase, and its putative metabolic role are further discussed.