Nucleotide sequence and expression in Escherichia coli of the cephalosporin acylase gene of a Pseudomonas strain.
The gene encoding cephalosporin acylase, which hydrolyzes 7-beta-(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA) to 7-aminocephalosporanic acid (7ACA) and glutaric acid, was cloned from a Pseudomonas sp. strain V22 and expressed in Escherichia coli, in a two-cistron system, and the enzyme was purified and characterized. The purified enzyme was composed of two non-identical subunits, their molecular weights were estimated by SDS-PAGE to be 40,000 and 22,000, and had a pI of 4.6. The amino acid sequence of the enzyme, deduced from the nucleotide sequence, showed high similarity (97%) with that of a previously reported acyI-encoded cephalosporin acylase. Cephalosporin acylase also resembles the bacterial gamma-glutamyl transpeptidases (GGTs) with respect to their molecular organization and amino acid sequence, but differs from them with respect to catalytic and immunological properties. Purified enzyme exhibited not only cephalosporin acylase activity, but also GGT activity. The Km values of the enzyme for GL-7ACA and L-gamma-glutamyl-p-nitroanilide were 6.1 and 3.8 mM, respectively. Cephalosporin acylase was not recognized by antibodies prepared against bacterial GGTs.
Ishiye
M
M
Product Development Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan.
Niwa
M
M
D000581:Amidohydrolases / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli; D008969:Molecular Sequence Data; D010399:Penicillin Amidase; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D005723:gamma-Glutamyltransferase / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D005723:gamma-Glutamyltransferase; D000581:Amidohydrolases; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase