OVEREXPRESSION OF THE NOCARDIA-LACTAMDURANS ALPHA-AMINOADIPYL-CYSTEINYL-VALINE SYNTHETASE IN STREPTOMYCES-LIVIDANS - THE PURIFIED MULTIENZYME USES CYSTATHIONINE AND 6-OXOPIPERIDINE 2-CARBOXYLATE AS SUBSTRATES FOR SYNTHESIS OF THE TRIPEPTIDE

Formation of the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D- valine (Aad-Cys-Val) is catalyzed by a multienzyme peptide synthetase encoded by the pcbAB gene in producers of beta-lactam antibiotics. The pcbAB gene of Nocardia lactamdurans was overexpressed in Streptomyces lividans giving a high Aad-Cys-Val synthetase activity. The synthetas e was purified 2785-foId to near homogeneity showing a molecular mass of 430 kDa by SDS/PAGE. The protein was identified in the gels with an tibodies to Aad-Cys-Val synthetase and by the formation of aminoacyl-s ynthetase thioester complex with [C-14]valine. The purified synthetase used a-aminoadipic acid or its lactam 6-oxopiperidine 2-carboxylic ac id but was unable to use piperideine 6-carboxylic acid or pipecolic ac id as substrates to form Aad-Cys-Val. L-Cystathionine, (2-amino-2-carb oxyethyl)-L-homocysteine, was used as substrate and formed Aad-Cys-Val with the same efficiency as L-cysteine. The product of the reaction e luted with authentic Aad-Cys-Val. The synthetase preparation was able to hydrolyze L-cystathionine by a pyridoxal-phosphate-independent mech anism which is not inhibited by propargylglycine, to form Aad-Cys-Val.