title: Thioesterase domain of delta-(l-alpha-Aminoadipyl)-l-cysteinyl-d-valine synthetase: alteration of stereospecificity by site-directed mutagenesis.
abstract: The carboxy-terminal thioesterase domain of delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine synthetase catalyzes the hydrolytic release of the tripeptide product (LLD-ACV). By site-directed mutagenesis an S3599A change was introduced into the highly conserved GXSXG motif, resulting in a more than 95 % decrease of penicillin production. Purification of the modified multienzyme showed surprisingly only a 50 % reduction of the peptide formation rate, with the stereoisomer delta-(l-alpha-aminoadipyl)-l-cysteinyl-l-valine (LLL-ACV) as the dominating product. Thioesterases of ACV synthetases differ from other thioesterases integrated in non-ribosomal peptide synthetases in their direct association with an epimerase domain, and their respective GXSXG-seryl residue is apparently not essential in acyl transfer leading to peptide release. Instead, this motif may be involved in the control of tripeptide epimerization by selection of the isomer to be released, and the construct supports the presence of LLL-ACV as an intermediate in penicillin biosynthesis.
authors.affiliation: Institute of Biochemistry and Molecular Biology, Technical University Berlin, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Kallow
authors.forename: J
authors.initials: J
authors.lastname: Kennedy
authors.forename: B
authors.initials: B
authors.lastname: Arezi
authors.forename: G
authors.initials: G
authors.lastname: Turner
authors.forename: H
authors.initials: H
authors.lastname: von Döhren
mesh_terms: D000215:Acylation; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001233:Aspergillus nidulans / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017124:Conserved Sequence / Q000235:genetics; D005285:Fermentation; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010406:Penicillins / Q000096:biosynthesis; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000235:genetics; D013237:Stereoisomerism; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D009842:Oligopeptides; D010406:Penicillins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C011638:5-(2-aminoadipyl)cysteinylvaline; D012694:Serine; D000255:Adenosine Triphosphate; D013869:Thiolester Hydrolases; D010453:Peptide Synthases; C095368:alpha-aminoadipyl-cysteinyl-valine synthetase