MOLECULAR CHARACTERIZATION OF THE ACYL-COENZYME-A - ISOPENICILLIN-N ACYLTRANSFERASE GENE (PENDE) FROM PENICILLIUM-CHRYSOGENUM AND ASPERGILLUS-NIDULANS AND ACTIVITY OF RECOMBINANT ENZYME IN ESCHERICHIA-COLI

The final step in the biosynthesis of β-lactam antibiotics in Penicillium chrysogenum and Aspergillus nidulans involves removal of the L-α-aminoadipyl side chain from isopenicillin N (IPN) and exchange with a nonpolar side chain. The enzyme catalyzing this reaction, acyl-coenzyme A:isopenicillin N acyltransferase (acyltransferase), was purified from P. chrysogenum and A. nidulans. Based on NH2-terminal amino acid sequence information, the acyltransferase gene (penDE) from P. chrysogenum and A. nidulans were cloned. In both organisms, penDE was located immediately downstream from the isopenicillin N synthetase gene (pcbC) and consisted of four exons encoding an enzyme of 357 amino acids (~ 40 kilodaltons [kDa]). The DNA coding sequences showed ~ 73% identity, while the amino acid sequences were ~ 76% identical. Noncoding DNA regions (including the region between pcbC and penDe) were not conserved. Acyltransferase activity from Escherichia coli producing the 40-kDa protein accepted either 6-aminopenicillanic acid or IPN as the substrate and made a penicillinase-sensitive antibiotic in the presence of phenylacetyl coenzyme A. Therefore, a single gene is responsible for converting IPN to penicillin G. The active form of the enzyme may result from processing of the 40-kDa monomeric precursor to a heterodimer containing subunits of 11 and 29 kDa.