SUBSTRATE-DEPENDENT ENANTIOSELECTIVITY OF A NOVEL HYDANTOINASE FROM ARTHROBACTER-AURESCENS DSM-3745 - PURIFICATION AND CHARACTERIZATION AS NEW MEMBER OF CYCLIC AMIDASES

A hydantoinase from Arthrobacter aurescens DSM 3745 has been purified to homogeneity with a yield of 77% using a three-step purification pro cedure. The active enzyme is a tetramer consisting of four identical s ubunits, each with a molecular mass of 49670 Da as determined by mass spectrometry. The N-terminal amino acid sequence of the enzyme indicat es sequence identities to cyclic amidases involved in the nucleotide m etabolism 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 catal ytic 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 hyda ntoinases 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 substr ate used for bioconversion: although it is strictly L-selective for th e cleavage of D,L-5-indolylmethylhydantoin, it appears to be D-selecti ve for the hydrolysis of D,L-methylthioethylhydantoin. Due to these fi ndings we conclude that this novel bacterial hydantoinase should be cl assified as a new member of the EC-group 3.5.2 of cyclic amidases. (C) 1998 Elsevier Science B.V. All rights reserved.