Thioesterase domain of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase: alteration of stereospecificity by site-directed mutagenesis

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• 作者：Kallow ; Wibke ; Kennedy ; Jonathan ; Arezi ; Bahram ; Turner ; Geoffrey ; von Dö ; hren ; Hans
• 关键词：thioesterase ; mutagenesis ; peptide synthetase ; biosynthesis ; stereospecificity ; ACV ; δ ; -(l-α ; -aminoadipyl)-l-cysteinyl-d-valine ; LLL-ACV ; δ ; -(l-α ; -aminoadipyl)-l-cysteinyl-l-valine ; DTE ; dithioerythreitol ; PPi ; inorganic pyrophosphate
• 刊名：Journal of Molecular Biology
• 出版年：2000
• 出版时间：March 24, 2000
• 年：2000
• 卷：297
• 期：2
• 页码：395-408
• 全文大小：326 K

文摘

The carboxy-terminal thioesterase domain of δ-(l-α-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 δ-(l-α-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.