Kaarin T. Mason

Novel Actinomycins Formed by Biosynthetic Incorporation of cis- and trans-4-Methylproline

Streptomyces parvulus (Streptomyces parvullus) normally produces actinomycin D; in the presence of cis-4-methylproline, this species synthesizes two additional actinomycins, designated K1c and K2c, in which one and two proline sites, respectively, are occupied by cis-4-methylproline. Analogously, actinomycins K1t and K2t are formed in the presence of trans-4-methylproline. Both mixtures were separated chromatographically, and the four novel actinomycins were obtained in crystalline form. Their biological activities were compared with that of actinomycin D in respect to inhibition of ribonucleic acid, deoxyribonucleic acid, and protein synthesis and antimicrobial potency. In all cases examined, the order of activity D > K1t > K1c > K2t > K2c was observed, and the same sequence prevailed in a spectroscopic measure of their binding to deoxyribonucleic acid. In addition, proton nuclear magnetic resonance studies revealed that the replacement of proline by cis-4-methylproline alters the conformation of the antibiotic molecule.

Identification of cis-5-methylproline in hydrolysates of actinomycin Z5

Abstract Actinomycins of the Z series, synthesized by Streptomyces fradiae , contain the unusual amino acid, N-methylalanine, but no proline. Hydrolysates of actinomycin Z5 were investigated using paper, gas and ion-exchange chromatographic procedures. Identification of an unknown amino acid in actinomycin Z5 as 5-methylproline was confirmed by mass spectrometry. Configuration of the imino acid was defined as cis .

3-Hydroxy-5-methylproline, a new amino acid identified as a component of actinomycin Z1

Abstract 3-Hydroxy-5-methylproline has been identified in hydrolysates of actinomycin Z 1 by ion-exchange and paper chromatography, paper electrophoresis, gas chromatography and mass spectrometry in comparison with the synthetic compound. The stereochemistry of this amino acid is under investigation. The amino acid composition of actinomycin Z 1 thus consists of threonine, hydroxythreonine, D-valine(2), 4-oxo-5-methylproline, 3-hydroxy-5-methylproline, sarcosine(2), N-methylalanine and N-methylvaline.

Biosynthetic studies with l-[2,3-3H2]valine as precursor of the d-valine moiety in actinomycin

Abstract The mechanism of inversion of l -valine to its d -enantiomer by Streptomyces parvulus was studied with l -[2,3- 3 H 2 ]valine as precursor. The data favor the view that there is a specific loss of the α-hydrogen of l -valine during the inversion and rule out a reaction mechanism in which α,β-dehydrovaline (valinyl) participates as an intermediate.

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

Actinomycins normally contain N-methyl-l-valine and either d-valine, d-alloisoleucine 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 (C1, C2, C3, E1, 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.

Further studies on the origin of sarcosine synthesized by streptomyces antibioticus and streptomyces chrysomallus

Abstract 1. 1. Evidence was obtained that sarcosine: (a) partially inhibits actinomycin formation by Streptomyces chrysomallus and Streptomyces antibiotics and (b) markedly inhibits the incorporation of proline, hydroxyproline and oxoproline into the antibiotic peptides. 2. 2. Experiments with (N-Me-14C)- and (1-14C)-labeled sarcosine have revealed that sarcosine is directly incorporated into the various actinomycins synthesized by S. chrysomallus and S. antibiotics. 3. 3. Radioisotope dilution experiments to establish a precursor-product relationship between glycine and sarcosine have proved to be inconclusive due to competition by the amino acids for entry into or exit from the cells. 4. 4. It is concluded that free or enzyme-bound glycine is first methylated and that the sarcosine formed is then incorporated into the actinomycin peptide.

Studies on the biological activities of actinomycins Z1 and Z5

Abstract The biological activities of actinomycins Z 1 , Z 5 , and IV (D) were compared. No consistent pattern was observed between the in vitro and in vivo results. Inhibition of E. coli DNA-dependent RNA polymerase in vitro followed the sequence IV > Z 1 > Z 5 ; however, for inhibition of RNA synthesis in B. subtilis and in HeLa cells the sequence was IV > Z 5 > Z 1 and the latter relationship was seen in the antimicrobial activity also. Physicochemical data did not agree with any of these findings. Difference spectra obtained with B. subtilis and M. lysodeikticus DNA followed the sequence Z 1 > IV > Z 5 , while the relative thermal denaturation profiles were the exact opposite, Z 5 > IV > Z 1 . These physicochemical criteria appear to be less reliable quantitative guides to biological activity. The relative ineffectiveness of actinomycin Z 1 in vivo is probably the result of permeability differences associated with the presence of an hydroxylated proline residue (3-hydroxy-4-oxo-5-methylproline).

Biogenetic origin of the D-isoleucine and N-methyl-L-alloisoleucine residues in the actinomycins.

Studies with 14C-labeled isoleucine stereisomers have established that l-alloisoleucine, d-alloisoleucine, and d-isoleucine may function as precursors for the biogenesis of d-isoleucine and N-methyl-l-alloisoleucine residues in actinomycin. l-[14C]isoleucine appears to be employed chiefly for d-alloisoleucine (and N-methylisoleucine [?] formation); however, its role in the biosynthesis of d-isoleucine and N-methylalloisoleucine remains unclear. The potential pathway of biosynthesis of d-isoleucine and N-methyl-l-isoleucine is discussed.