Acyl-coenzyme A: isopenicillin N acyltransferase from Penicillium chrysogenum: effect of amino acid substitutions at Ser227, Ser230 and Ser309 on proenzyme cleavage and activity
| Autor: | James R. Miller, Jack E. Baldwin, Matthew B. Tobin, John D. Sutherland, Stephen C.J. Cole, Steven Kovacevic |
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| Rok vydání: | 1994 |
| Předmět: |
Stereochemistry
Molecular Sequence Data Biology Penicillium chrysogenum Cleavage (embryo) Microbiology Acylation chemistry.chemical_compound Structure-Activity Relationship Biosynthesis Genetics Escherichia coli Serine Penicillin-Binding Proteins Amino Acid Sequence Molecular Biology Peptide sequence Conserved Sequence chemistry.chemical_classification Enzyme Precursors Base Sequence Recombinant Proteins Amino acid Enzyme chemistry Biochemistry Acyltransferase Mutagenesis Site-Directed Protein Processing Post-Translational Acyltransferases Cysteine |
| Zdroj: | FEMS microbiology letters. 121(1) |
| ISSN: | 0378-1097 |
| Popis: | Using a high level Escherichia coli expression system for the Penicillium chrysogenum penDE gene, we have produced acyl-coenzyme A: isopenicillin N acyltransferase (AT) enzymes containing amino acid substitutions at three conserved Ser residues. Chosen for study based on amino acid sequence homologies to other proteins, Ser227, Ser230 and Ser309 were changed to Cys or Ala to assess amino acid side chain involvement in proenzyme cleavage and AT enzyme mechanism. Substitutions at Ser230 had no effect on proenzyme cleavage, acyl-coenzyme A: IPN acyltransferase (IAT) or acyl-coenzyme A:6-aminopenicillanic acid acyltransferase (AAT) activities. While Ser227--Cys had no effect, Ser227--Ala produced uncleaved proenzyme lacking both AAT and IAT activities, suggesting that the presence of a nucleophilic side chain at this residue is required for proenzyme cleavage and AT activity. Substitution of Ser309--Cys did not appreciably prevent proenzyme cleavage, IAT or AAT activity. Recombinant AT (recAT) proenzyme containing Ser309--Ala was cleaved; however, IAT and AAT activities were not observed. This separation of proenzyme cleavage from IAT and AAT activities has not been previously observed, and suggests that Ser309 is involved in substrate acylation. |
| Databáze: | OpenAIRE |
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