Abstract
A hydantoinase from Arthrobacter aurescens DSM 3745 has been purified to
homogeneity with a yield of 77\% using a three-step purification
procedure. The active enzyme is a tetramer consisting of four identical
subunits, each with a molecular mass of 49670 Da as determined by mass
spectrometry. The N-terminal amino acid sequence of the enzyme indicates
sequence identities to cyclic amidases involved in the nucleotide
metabolism as the D-hydantoinase from Agrobacterium radiobacter (53\%),
the D-selective dihydropyrimidinase from Bacillus stearothermophilus
(38\%), the allantoinase from Rana catesbeiana (26\%), as well as to the
catalytic subunit of the urease from Heliobacter pylori (50\%). However,
all studies based on substrate-dependent growth, induction and catalytic
behavior documented the novelty of the bacterial hydantoinase and that
its physiological role is not related to any of these enzymes or known
metabolic pathways. Its substrate specificity differs from hydantoinases
listed in Enzyme Nomenclature and is rather more predominant for the
cleavage of aryl-than for alkyl-hydantoin, derivatives. It is shown that
the stereoselectivity of this enzyme depends on the substrate used for
bioconversion: although it is strictly L-selective for the cleavage of
D,L-5-indolylmethylhydantoin, it appears to be D-selective for the
hydrolysis of D,L-methylthioethylhydantoin. Due to these findings we
conclude that this novel bacterial hydantoinase should be classified as
a new member of the EC-group 3.5.2 of cyclic amidases. (C) 1998 Elsevier
Science B.V. All rights reserved.
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