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
Jjr. Coque et al., 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, European journal of biochemistry, 242(2), 1996, pp. 264-270
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.