Branched-Chain Amino Acid Substitutions in the Biosynthesis of the Antibiotic Actinomycin

Abstract

Actinomycins normally contain N-methyl-L-valine and either D-valine, Dalloisoleucine or both amino acids in the molecule. During antibiotic formation in a medium supplemented with one of the four isoleucine stereoisomers, Streptomyces parvulus and S. chrysomallus form complex actinomycin mixtures (Cl, C2, C3, El, and E2-like compounds). Although chromatographic techniques suggested that single homogeneous components had been isolated, subsequent studies indicated that such chromatographic fractions probably consisted of multiple isomers of actinomycin. Amino acid analyses revealed the presence of N-methylvaline and/or N-methylalloisoleucine and, in addition, D-isoleucine, D-valine, and D-alloisoleucine were frequently found in a given fraction.Schmidt-Kastner (30, 31) first noted that new actinomycins (El and E2) were synthesized by Streptomyces chrysomallus when the organism was grown in the presence of DL-isoleucine. It was reported (30, 31) that one or two residues of N-methylisoleucine replaced one or two residues of N-methylvaline, respectively, in the parent compound, actinomycin C3. More recently, our laboratory demonstrated that S. antibioticus and S. chrysomallus both form actinomycin mixtures which contain appreciable amounts of D-isoleucine and N-methyl-Lalloisoleucine (rather than N-methyl-isoleucine) when synthesis occurs with an isoleucine stereoisomer (15,35). N-methyl-L-alloisoleucine and D-isoleucine substitute for N-methyl-Lvaline and either D-valine or D-alloisoleucine, respectively, in the antibiotic peptides. Individual biosynthetic actinomycins were not examined; therefore, we initiated studies to establish the branched-chain amino acid composition of the isoleucine-induced actinomycin components formed by S. chrysomallus and S. parvulus. MATERIALS AND METHODSOrganisms and conditions of cultivation. S. chrysomallus (ATCC 11523) and S. parvulus (ATCC 12434) were maintained on slants of glucose-yeast extract-malt extract agar medium (9). Incubation was for 3 to 5 days at 30 C until sporulation had occurred; slants were then kept at 4 C until further use.For production of actinomycin the organisms were

Biogenetic Origin of the d -Isoleucine and N -Methyl- l -Alloisoleucine Residues in the Actinomycins

“…The concentration of actinomycin was determined spectrophotometrically (23). Determination of amino acids was accomplished with the Beckman Spinco automatic amino acid analyzer (model 120C) (38,39) or by a modification of the paper chromatographic procedure described by Naftalin (23,29). Optical configuration of amino acids was established with dialyzed prepa-226 YAJIMA, MASON, AND KATZ rations of L-or 1-amino acid oxidase (23,25,30).…”

“…Separation of amino acids in hydrolysates was achieved by high-voltage electrophoresis (23,38,39). Amino acid standards were also applied to locate the amino acids.…”

“…Streptomyces antibioticus (ATCC 14888), Streptomyces chrysomallus (ATCC 11523), and Streptomyces parvulus (ATCC 12434) were cultivated on slants of glucose-yeast extract-malt extract agar medium (15). For production of actinomycin, an organism was grown first in NZ amine medium for 48 h; synthesis of actinomycin mixtures was effected subsequently in glutamic acid-galactose-glucose or fructose-glutamic acid-histidine medium as described previously (19,21,23,39).…”

“…The antibiotic synthesized by S. antibioticus or S. parvulus was hydrolyzed in 6 N HCl as detailed elsewhere (23). In most experiments, S. chrysomallus mixtures were first separated into individual components by descending paper chromatography and then purified by the procedure of Yajima et al (39) prior to acid hydrolysis.…”

“…i-Isoleucine and N-methyl-L-alloisoleucine were identified as amino acid constituents of the isoleucine-induced antibiotics elaborated by various Streptomyces species (20,38,39). The observation that D-isoleucine is a constituent of certain monamycin congeners constituted the first report concerning an exception to the rule of a-epimerization (4,5,7).…”