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

Eur J Biochem. 1996 Dec 1;242(2):264-70. doi: 10.1111/j.1432-1033.1996.0264r.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Gel
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • Cloning, Molecular / methods
  • Cystathionine / metabolism*
  • Kinetics
  • Multienzyme Complexes / biosynthesis
  • Multienzyme Complexes / isolation & purification
  • Multienzyme Complexes / metabolism
  • Nocardia / enzymology*
  • Nocardia / genetics
  • Peptide Synthases / biosynthesis
  • Peptide Synthases / isolation & purification
  • Peptide Synthases / metabolism*
  • Pipecolic Acids / metabolism*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Restriction Mapping
  • Streptomyces / metabolism*
  • Substrate Specificity
  • Ultrafiltration

Substances

  • 6-oxopiperidine-2-carboxylic acid
  • Multienzyme Complexes
  • Pipecolic Acids
  • Recombinant Proteins
  • Cystathionine
  • Peptide Synthases
  • alpha-aminoadipyl-cysteinyl-valine synthetase