title: Amino-acid substitutions in the cleavage site of acyl-coenzyme A:isopenicillin N acyltransferase from Penicillium chrysogenum: effect on proenzyme cleavage and activity.
abstract: Site-directed mutagenesis of the penDE gene and expression in Escherichia coli has produced recombinant acylcoenzyme A:isopenicillin N acyltransferase (re-AT) containing amino-acid substitutions in the proenzyme cleavage site (decreases) region (Asp-Gly102 decreases Cys103-Thr-Thr). The effect of these substitutions on proenzyme cleavage and AT activity has been investigated. The re-AT with substitutions at Cys103 (Cys103-->Ser, Cys103-->Ala and Cys103-->Trp) were uncleaved and inactive. Substitutions at Asp101 and Gly102 (Asp101-->Gly, Gly102-->Ala, Gly102-->Val, Gly102-->Met, Gly102-->Val and Asp101Gly102-->GlyPhe) did not prevent proenzyme cleavage or abolish AT activity. Thr105-->Ser and Thr105-->Ala substitutions did not prevent proenzyme cleavage or AT activity; however, AT containing Thr105-->Val resulted in a significant inhibition of proenzyme cleavage.
authors.affiliation: Dyson Perrins Laboratory, Oxford, UK.
authors.forename: M B
authors.initials: MB
authors.lastname: Tobin
authors.forename: S C
authors.initials: SC
authors.lastname: Cole
authors.forename: J R
authors.initials: JR
authors.lastname: Miller
authors.forename: J E
authors.initials: JE
authors.lastname: Baldwin
authors.forename: J D
authors.initials: JD
authors.lastname: Sutherland
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D046915:Penicillin-Binding Proteins; D010408:Penicillium chrysogenum / Q000201:enzymology* / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D004792:Enzyme Precursors; D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins; D000217:Acyltransferases; C018756:acyl-CoA-6-aminopenicillanic acid acyltransferase