Combined purification of actinomycin synthetase I and 3-hydroxyanthranilic acid 4-methyltransferase from Streptomyces antibioticus.
Actinomycin synthetase I, which activates the precursor of the chromophore of actinomycin, has been purified nearly 1000-fold from extracts of Streptomyces antibioticus. The enzyme has an M(r) of 45,000 and appears to function as a single polypeptide chain. The formation of the enzyme is repressed when S. antibioticus mycelium is grown on glucose as a carbon source. Several benzoic acid derivatives, including intermediates in the putative pathway for actinomycin biosynthesis, were capable of supporting ATP-pyrophosphate exchange catalyzed by actinomycin synthetase I. Interestingly, three synthetic phenoxaziones also functioned as substrates in the exchange reaction. It proved possible to purify a second enzyme involved in actinomycin biosynthesis, 3-hydroxyanthranilic acid 4-methyltransferase, from the same extracts prepared for the purification of actinomycin synthetase I and by similar procedures. Streptomyces lividans, previously shown to contain a silent gene for the enzyme phenoxazione synthase, apparently does not contain a silent pathway for the complete synthesis of actinomycin since neither actinomycin synthetase I nor the methyltransferase could be detected in strains containing a cloned sequence that activates phenoxazione synthase expression.
Jones
G H
GH
Department of Biology, Emory University, Atlanta, Georgia 30322.
D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008780:Methyltransferases / Q000302:isolation & purification*; D009713:Nucleotidyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D010078:Oxazines / Q000494:pharmacology; D013303:Streptomyces antibioticus / Q000201:enzymology*; D013379:Substrate Specificity
D010078:Oxazines; C020456:phenoxazinone; C055341:3-hydroxyanthranilate-4-methyltransferase; D008780:Methyltransferases; D009713:Nucleotidyltransferases; C075125:actinomycin synthetase I