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

, , , and . JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, 6 (3): 387-397 (March 1999)
DOI: {10.1016/S1381-1177(98)00137-4}


A D-specific hydantoinase has been purified to homogeneity from Arthrobacter crystallopoietes DSM 20117 with a yield of 5\% related to the crude extract. 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 with the metal-chelating agent EDTA had no inhibitory effect, while 8-hydroxyquinoline-5-sulfonic acid resulted in a complete and irreversible inactivation. The purified enzyme contains zinc as cofactor, which could be detected by subjection to direct analysis using inductive/coupled plasma-atomic emission spectrometry. The hydantoinase has a wide substrate specificity for the D-selective cleavage of 5-monosubstituted hydantoin derivatives with aliphatic 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 substrate (V-max = 35 U/mg). The N-terminal amino acid sequence of the enzyme shows distinct homologies to other metal-dependent cyclic amidases involved in the nucleotide metabolism especially to dihydropyrimidinases as well as to ureases, L- and unselective hydantoinases. Due to these findings, this enzyme 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.

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