A novel N-carbamoyl-L-amino acid amidohydrolase of Pseudomonas sp. strain ON-4a: purification and characterization of N-carbamoyl-L-cysteine amidohydrolase expressed in Escherichia coli.
N-carbamoyl-L-cysteine amidohydrolase (NCC amidohydrolase) was purified and characterized from the crude extract of Escherichia coli in which the gene for NCC amidohydrolase of Pseudomonas sp. strain ON-4a was expressed. The enzyme was purified 58-fold to homogeneity with a yield of 16.1% by three steps of column chromatography. The results of gel filtration on Sephacryl S-300 and SDS-polyacrylamide gel electrophoresis suggested that the enzyme was a tetramer protein of identical 45-kDa subunits. The optimum pH and temperature of the enzyme activity were pH 9.0 and 50 degrees, respectively. The enzyme required Mn(2+) ion for activity expression and was inhibited by EDTA, Hg(2+) and sulfhydryl reagents. The enzyme was strictly specific for the L-form of N-carbamoyl-amino acids as substrates and exhibited high activity in the hydrolysis of N-carbamoyl-L-cysteine as substrate. These results suggested that the NCC amidohydrolase is a novel L-carbamoylase, different from the known L-carbamoylases.
Ohmachi
Tetsuo
T
Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki 036-8561, Japan. tohmachi@cc.hirosaki-u.ac.jp
Narita
Megumi
M
Kawata
Maki
M
Bizen
Akiko
A
Tamura
Yoshiharu
Y
Asada
Yoshihiro
Y
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002845:Chromatography; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008345:Manganese / Q000494:pharmacology; D008628:Mercury / Q000494:pharmacology; D008970:Molecular Weight; D021122:Protein Subunits; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D003067:Coenzymes; D004791:Enzyme Inhibitors; D021122:Protein Subunits; D011994:Recombinant Proteins; D008345:Manganese; D004492:Edetic Acid; D000581:Amidohydrolases; C085345:1-carbamoyl-L-amino acid amidohydrolase; D008628:Mercury