Hydantoin racemase from Arthrobacter aurescens DSM 3747: heterologous expression, purification and characterization

In Arthrobacter aurescens DSM 3747 three enzymes are involved in the comple te conversion of slowly racemizing 5'-monosubstituted D,L-hydantoins to L-a mino acids, a stereoselective hydantoinase, a stereospecific L-N-carbamoyla se and a hydantoin racemase. The gene encoding the hydantoin racemase, desi gnated hyuA, was identified upstream of the previously described L-N-carbam oylase gene in the plasmid pAW16 containing genomic DNA of A. aurescens. Th e gene hyuA which encodes a polypeptide of 25.1 kDa, was expressed in Esche richia coli and the recombinant protein purified to homogeneity and further characterized. The optimal condition for racemase activity were pH 8.5 and 55 degrees C with L-5-benzylhydantoin as substrate. The enzyme was complet ely inhibited by HgCl2 and iodoacetamide and stimulated by addition of dith iothreitol. No effect on enzyme activity was seen with EDTA. The enzyme sho wed preference for hydantoins with arylalkyl side chains. Kinetic studies r evealed substrate inhibition towards the aliphatic substrate L-5-methylthio ethylhydantoin. Enzymatic racemization of D-5-indolylmethylenehydantoin in D2O and NMR analysis showed that the hydrogen at the chiral center of the h ydantoin is exchanged against solvent deuterium during the racemization. (C ) 2000 Elsevier Science B.V. All rights reserved.