Open AccessAcyl‐coenzyme A: isopenicillin N acyltransferase from Penicillium chrysogenum : effect of amino acid substitutions at Ser 227 , Ser 230 and Ser 309 on proenzyme cleavage and activity
Author(s) -
Tobin Matthew B.,
Cole Stephen C.J.,
Kovacevic Steven,
Miller James R.,
Baldwin Jack E.,
Sutherland John D.
Publication year - 1994
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1994.tb07073.x
Subject(s) - penicillium chrysogenum , chemistry , biochemistry , stereochemistry , acyltransferase , amino acid , enzyme , coenzyme a , reductase
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, Ser 227 , Ser 230 and Ser 309 were changed to Cys or Ala to assess amino acid side chain involvement in proenzyme cleavage and AT enzyme mechanism. Substitutions at Ser 230 had no effect on proenzyme cleavage, acyl‐coenzyme A: IPN acyltransferase (IAT) or acyl‐coenzyme A: 6‐aminopenicillanic acid acyltransferase (AAT) activities. While Ser 227 →Cys had no effect, Ser 227 →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 Ser 309 →Cys did not appreciably prevent proenzyme cleavage, IAT or AAT activity. Recombinant AT (recAT) proenzyme containing Ser 309 →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 Ser 309 is involved in substrate acylation.