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 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 [14C]valine. The purified synthetase used alpha-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-Cystathionine, (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.
Coque
J J
JJ
Faculty of Biology, Department of Ecology, Genetics and Microbiology, University of León, Spain.
de la Fuente
J L
JL
Liras
P
P
Martín
J F
JF
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular / Q000379:methods; D003540:Cystathionine / Q000378:metabolism*; D007700:Kinetics; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D009615:Nocardia / Q000201:enzymology* / Q000235:genetics; D010453:Peptide Synthases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010875:Pipecolic Acids / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013302:Streptomyces / Q000378:metabolism*; D013379:Substrate Specificity; D014462:Ultrafiltration
C030878:6-oxopiperidine-2-carboxylic acid; D009097:Multienzyme Complexes; D010875:Pipecolic Acids; D011994:Recombinant Proteins; D003540:Cystathionine; D010453:Peptide Synthases; C095368:alpha-aminoadipyl-cysteinyl-valine synthetase