title: The isopenicillin-N acyltransferase of Penicillium chrysogenum has isopenicillin-N amidohydrolase, 6-aminopenicillanic acid acyltransferase and penicillin amidase activities, all of which are encoded by the single penDE gene.
abstract: The isopenicillin-N acyltransferase of Penicillium chrysogenum catalyzes the conversion of the biosynthetic intermediate isopenicillin N to the hydrophobic penicillins. The isopenicillin-N acyltransferase copurified with the acyl-CoA:6-aminopenicillanic acid (6-APA) acyltransferase 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 sequence similar to that of the active site of thioesterases was found in the isopenicillin-N acyltransferase, suggesting that this site is involved in the transfer of phenylacetyl residues from phenylacetyl thioesters. Purified isopenicillin-N acyltransferase also showed isopenicillin-N amidohydrolase, penicillin transacylase and penicillin amidase activities. 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 converted into benzylpenicillin. Penicillin transacylase exchanged side chains between two hydrophobic penicillin molecules; or between one penicillin molecule and 6-APA. The penicillin amidase activity is probably the reverse of the biosynthetic acyl-CoA:6-APA acyltransferase. Four P. chrysogenum mutants deficient in acyl-CoA:6-APA acyltransferase lacked the other four related activities. Transformation of these mutants with the penDE gene restored all five enzyme activities.
authors.affiliation: Department of Ecology, Genetics and Microbiology, Faculty of Biology, University of Leon, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Alvarez
authors.forename: B
authors.initials: B
authors.lastname: Meesschaert
authors.forename: E
authors.initials: E
authors.lastname: Montenegro
authors.forename: S
authors.initials: S
authors.lastname: Gutiérrez
authors.forename: B
authors.initials: B
authors.lastname: Díez
authors.forename: J L
authors.initials: JL
authors.lastname: Barredo
authors.forename: J F
authors.initials: JF
authors.lastname: Martín
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000581:Amidohydrolases / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D003470:Culture Media; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D015870:Gene Expression; D008969:Molecular Sequence Data; D009154:Mutation; D010399:Penicillin Amidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism*; D010408:Penicillium chrysogenum / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology
chemical_list: D003470:Culture Media; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D013439:Sulfhydryl Reagents; D000217:Acyltransferases; C018756:acyl-CoA-6-aminopenicillanic acid acyltransferase; D000581:Amidohydrolases; D010399:Penicillin Amidase; C006414:penicillin N; D004229:Dithiothreitol