Purification and characterization of actinomycin synthetase I, a 4-methyl-3-hydroxyanthranilic acid-AMP ligase from Streptomyces chrysomallus.
Actinomycin synthetase I was purified to homogeneiety from actinomycin-producing Streptomyces chrysomallus. The purified enzyme is a single polypeptide chain of M(r) 45,000. It catalyzes the formation of the adenylate of 4-methyl-3-hydroxyanthranilic acid (4-MHA) from the free acid and ATP in an equilibrium reaction. 4-MHA is the precursor of the chromophoric part of actinomycin. By using the 4-MHA analogue, 4-methyl-3-hydroxybenzoic acid, as a model substrate it could be established that the equilibrium constant Keq is independent on enzyme concentration, which suggests that no stoichiometric acyladenylate-enzyme complex is formed in contrast to observations made with aminoacyl adenylates formed by aminoacyl-tRNA synthetases or multifunctional peptide synthetases. Actinomycin synthetase I does not charge itself with substrate carboxylic acid via a covalent thioester bond as is usual for amino acid activation in non-ribosomal peptide synthesis. In addition, the enzyme does not act as an acyl-coenzyme A ligase as revealed by its inability to release AMP in the presence of 4-MHA or other structurally related aromatic carboxylic acids, coenzyme A and ATP. Additional analysis of the activation reaction showed that it is exothermic, whereas the free enthalpy change delta G0 is positive due to a negative entropy change indicating a strong influence of restriction of random motion on the course of the reaction. Determinations of Km and kcat of various substrate carboxylic acids revealed the highest kcat/Km ratio for the natural substrate 4-MHA. From these properties, actinomycin synthetase I represents the prototype of novel chromophore activating enzymes involved in non-ribosomal synthesis of chromopeptide lactones in streptomycetes.
Keller
U
U
Institut für Biochemie und Molekulare Biologie, Technische Universität Berlin, Federal Republic of Germany.
Schlumbohm
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W
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D062367:ortho-Aminobenzoates / Q000378:metabolism*
D062367:ortho-Aminobenzoates; C034508:4-methyl-3-hydroxyanthranilic acid; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; C075125:actinomycin synthetase I