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 beta-lactam antibiotics in Penicillium chrysogenum and Aspergillus nidulans involves removal of the L-alpha-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 (approximately 40 kilodaltons [kDa]). The DNA coding sequences showed approximately 73% identity, while the amino acid sequences were approximately 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.
Tobin
M B
MB
Department of Molecular Genetics Research, Lilly Research Laboratories, Indianapolis, Indiana 46285.
Fleming
M D
MD
Skatrud
P L
PL
Miller
J R
JR
D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005800:Genes, Fungal; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D046915:Penicillin-Binding Proteins; D010408:Penicillium chrysogenum / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins; D000217:Acyltransferases; C018756:acyl-CoA-6-aminopenicillanic acid acyltransferase