THE ISOPENICILLIN-N ACYLTRANSFERASE OF PENICILLIUM-CHRYSOGENUM HAS ISOPENICILLIN-N AMIDOHYDROLASE, 6-AMINOPENICILLANIC ACID ACYLTRANSFERASEAND PENICILLIN AMIDASE ACTIVITIES, ALL OF WHICH ARE ENCODED BY THE SINGLE PENDE GENE

The isopenicillin-N acyltransferase of Penicillium chrysogenum catalyz es the conversion of the biosynthetic intermediate isopenicillin N to the hydrophobic penicillins. The isopenicillin-N acyltransferase copur ified with the acyl-CoA:6-aminopenicillanic acid (6-APA) acyltransfera se activity which transfers an acyl residue from acyl-CoA derivatives (e.g. phenylacetyl-CoA, phenoxyacetyl-CoA) to 6-APA. Other thioesters of phenylacetic acid were also used as substrates. An amino acid seque nce similar to that of the active site of thioesterases was found in t he isopenicillin-N acyltransferase, suggesting that this site is invol ved in the transfer of phenylacetyl residues from phenylacetyl thioest ers. Purified isopenicillin-N acyltransferase also showed isopenicilli n-N amidohydrolase, penicillin transacylase and penicillin amidase act ivities. The isopenicillin-N amidohydrolase (releasing 6-APA) showed a much lower specific activity than the isopenicillin-N acyltransferase of the same enzyme preparation, suggesting that in the isopenicillin- N acyltransferase reaction the 6-APA is not released and is directly c onverted into benzylpenicillin. Penicillin transacylase exchanged side chains between two hydrophobic penicillin molecules; or between one p enicillin molecule and 6-APA. The penicillin amidase activity is proba bly the reverse of the biosynthetic acyl-CoA:6-APA acyltransferase. Fo ur P. chrysogenum mutants deficient in acyl-CoA:6-APA acyltransferase lacked the other four related activities. Transformation of these muta nts with the penDE gene restored all five enzyme activities.