Thioesterase domain of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase: Alteration of stereospecificity by site-directed mutagenesis

The carboxy-terminal thioesterase domain of delta-(L-alpha-aminoadipyl)-L-c ysteinyl-D-valine synthetase catalyzes the hydrolytic release of the tripep tide product (LLD-ACV). By site-directed mutagenesis an S3599A change was i ntroduced into the highly conserved GXSXG motif, resulting in a more than 9 5% decrease of penicillin production. Purification of the modified multienz yme showed surprisingly only a 50% reduction of the peptide formation rate, with the stereoisomer delta-(L-alpha-aminoadipyl)-L-cysteinyl-L-valine (LL L-ACV) as the dominating product. Thioesterases of ACV synthetases differ f rom other thioesterases integrated in non-ribosomal peptide synthetases in their direct association with an epimerase domain, and their respective GXS XG-seryl residue is apparently not essential in acyl transfer leading to pe ptide release. Instead, this motif may be involved in the control of tripep tide epimerization by selection of the isomer to be released, and the const ruct supports the presence of LLL-ACV as an intermediate in penicillin bios ynthesis. (C) 2000 Academic Press.