Overexpression of the Nocardia lactamdurans α‐Aminoadipyl‐Cysteinyl‐Valine Synthetase in Streptomyces lividans

Formation of the tripeptide δ‐( l ‐α‐aminoadipyl)‐ l ‐cysteinyl‐ d ‐valine (Aad‐Cys‐Val) is catalyzed by a multienzyme peptide synthetase encoded by the pcbAB gene in producers of β‐lactam antibiotics. The pcbAB gene of Nocardia Lactamdurans was overexpressed in Streptomyces Lividans giving a high Aad‐Cys‐Val synthetase activity. The synthetase was purified 2785‐fold to near homogeneity showing a molecular mass of 430 kDa by SDS/PAGE. The protein was identified in the gels with antibodies to Aad‐Cys‐Val synthetase and by the formation of aminoacyl‐synthetase thioester complex with [ 14 C]valine. The purified synthetase used α‐aminoadipic acid or its lactam 6‐oxopiperidine 2‐carboxylic acid but was unable to use piperideine 6‐carboxylic acid or pipecolic acid as substrates to form Aad‐Cys‐Val. l ‐Cysta‐thionine, (2‐amino‐2‐carboxyethyl)‐ l ‐homocysteine, was used as substrate and formed Aad‐Cys‐Val with the same efficiency as l ‐cysteine. The product of the reaction eluted with authentic Aad‐Cys‐Val. The synthetase preparation was able to hydrolyze l‐ cystathionine by a pyridoxal‐phosphate‐independent mechanism which is not inhibited by propargylglycine, to form Aad‐Cys‐Val.