Hydantoin racemase from Arthrobacter aurescens DSM 3747: heterologous expression, purification and characterization.
In Arthrobacter aurescens DSM 3747 three enzymes are involved in the complete conversion of slowly racemizing 5'-monosubstituted D,L-hydantoins to L-amino acids, a stereoselective hydantoinase, a stereospecific L-N-carbamoylase and a hydantoin racemase. The gene encoding the hydantoin racemase, designated hyuA, was identified upstream of the previously described L-N-carbamoylase gene in the plasmid pAW16 containing genomic DNA of A. aurescens. The gene hyuA which encodes a polypeptide of 25.1 kDa, was expressed in Escherichia 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 completely inhibited by HgCL2 and iodoacetamide and stimulated by addition of dithiothreitol. No effect on enzyme activity was seen with EDTA. The enzyme showed preference for hydantoins with arylalkyl side chains. Kinetic studies revealed substrate inhibition towards the aliphatic substrate L-5-methylthioethylhydantoin. Enzymatic racemization of D-5-indolylmethylenehydantoin in D2O and NMR analysis showed that the hydrogen at the chiral center of the hydantoin is exchanged against solvent deuterium during the racemization.
Wiese
A
A
Institut für Industrielle Genetik, Universität Stuttgart, Germany.
Pietzsch
M
M
Syldatk
C
C
Mattes
R
R
Altenbuchner
J
J
D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002614:Chelating Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D017666:Deuterium Oxide / Q000493:pharmacokinetics; D004202:Disinfectants / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006827:Hydantoins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008627:Mercuric Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D019998:Racemases and Epimerases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D013867:Thiohydantoins / Q000378:metabolism
D001426:Bacterial Proteins; D002614:Chelating Agents; D004202:Disinfectants; D004791:Enzyme Inhibitors; D006827:Hydantoins; D013867:Thiohydantoins; C031427:5-benzylhydantoin; D008627:Mercuric Chloride; C023447:methylthiohydantoin; D004492:Edetic Acid; D019998:Racemases and Epimerases; C074773:hydantoin racemase; D017666:Deuterium Oxide; D004229:Dithiothreitol; D007460:Iodoacetamide