Abstract
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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