A D-specific hydantoin amidohydrolase: properties of the metalloenzyme purified from Arthrobacter crystallopoietes

A D-specific hydantoinase has been purified to homogeneity from Arthrobacte r crystallopoietes DSM 20117 with a yield of 5% related to the crude extrac t. The active enzyme is a tetramer of 257 kDa consisting of four identical subunits, each with a molecular mass of 60 kDa. Incubation of the enzyme wi th the metal-chelating agent EDTA had no inhibitory effect, while 8-hydroxy quinoline-5-sulfonic acid resulted in a complete and irreversible inactivat ion. The purified enzyme contains zinc as cofactor, which could be detected by subjection to direct analysis using inductive/coupled plasma-atomic emi ssion spectrometry. The hydantoinase has a wide substrate specificity for t he D-selective cleavage of 5-monosubstituted hydantoin derivatives with ali phatic and aromatic side chains. The V-max-value for phenylhydantoin is 217 U/mg, the K-m-value is 8 mM. Dihydrouracil was found to be a natural subst rate (V-max = 35 U/mg). The N-terminal amino acid sequence of the enzyme sh ows distinct homologies to other metal-dependent cyclic amidases involved i n the nucleotide metabolism especially to dihydropyrimidinases as well as t o ureases, L- and unselective hydantoinases. Due to these findings, this en zyme has to be considered as a possible link in the evolution to related L- selective and unselective hydantoinases from the genus of Arthrobacter. (C) 1999 Elsevier Science B.V. All rights reserved.