Enzymatic characterisation of the multifunctional enzyme δ‐(l‐α‐aminoadipyl)‐l‐cysteinyl‐d‐valine synthetase from Streptomyces clavuligerus

• Published in: European journal of biochemistry Vol. 205; no. 2; pp. 687 - 694
• Main Authors: SCHWECKE, Torsten, AHARONOWITZ, Yair, PALISSA, Harriet, DÖHREN, Hans, KLEINKAUF, Horst, LIEMPT, Henk
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 15.04.1992; Blackwell
• Subjects: Amino Acyl-tRNA Synthetases - metabolism; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, Gel; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Kinetics; Ligases; Molecular Weight; Peptide Synthases - isolation & purification; Peptide Synthases - metabolism; Streptomyces - enzymology; Ligase; Streptomycetaceae; Multienzyme complex; Enzyme; Molecular weight determination; Streptomyces clavuligerus; Actinomycetales; Enzymatic activity; Bacteria; Actinomycetes; Kinetic parameter; ATP; Physicochemical properties
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1992.tb16830.x

δ‐(l‐α‐Aminoadipyl)‐l‐cysteinyl‐d‐valine (ACV) synthetase, the multienzyme catalyzing the formation of ACV from the constituent amino acids and ATP in the presence of Mg2+ and dithioerythritol, was purified about 2700‐fold from Streptomyces clavuligerus. The molecular mass of the native enzyme as determined by gel filtration chromatography is 560 kDa, while that determined by denaturing gel electrophoresis is 500 kDa. The enzyme is able to catalyze pyrophosphate exchange in dependence on l‐cysteine and l‐ and l‐valine, but no l‐α‐aminoadipic‐acid‐dependent ATP/PPi exchange could be detected. Other l‐ and l‐cysteine‐ and l‐valine‐activating enzymes present in crude extracts were identified as aminoacyl‐tRNA synthetases which could be separated from ACV synthetase. The molecular mass of these enzymes is 140 kDa for l‐valine ligase and 50 kDa for l‐cysteine ligase. The dissociation constants have been estimated, assuming three independent activation sites, to be 1.25 mM and 1.5 mM for cysteine and ATP, and 2.4 mM and 0.25 mM for valine and ATP, respectively. The enzyme forms a thioester with α‐aminoadipic acid and with valine in a molar ratio of 0.6:1 (amino acid/enzyme). Thus, the bacterial ACV synthetase is a multifunctional peptide synthetase, differing from fungal ACV synthetases in its mechanism of activation of the non‐protein amino acid.