Biological studies on mikamycin. II. Laboratory investigations of mikamycin A and mikamycin B.
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Commercial preparations of mikamycin have been shown to act as both inhibitors of mitochondrial protein synthesis and respiration. These preparations are shown to consist of two major streptogramin components (mikamycin A and mikamycin B) and a number of minor components. The major streptogramin components which inhibit mitochondrial protein synthesis in vitro are without effect in vivo due to whole cell impermeability to these compounds. A minor antimycin A-like component is the active compound in mikamycin preparations which inhibits growth of yeast cells on ethanol. The site of this inhibition is at the level of respiratory Comples III. The mitochondrial [mik 1-r] mutation confers resistance to this minor growth inhibitory component and cross resistance to antimycin A. For clarity the designation mik 1 has therefore been renamed ana 1 to denote the mitochondrial determinant conferring resistance to antimycin A. Genetic and physical mapping studies localise the ana 1 determinant in the region of mitochondrial DNA specifying cytochrome b. It is proposed that the ana 1 locus is part of a gene specifying a membrane component of Complex III.
In mikamycin B fermentation, some procedures were examined to remove the participation of mikamycin B lactonase, which reduces mikamycin B titers. Addition of enzyme inhibitors and control of pH resulted in the elimination of the enzyme activity, and in the stimulation of antibiotic production.
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A 3-hydroxypicolinic acid activating enzyme from etamycin producing Streptomyces griseoviridus has been purified to apparent homogeneity. Etamycin is a member of mikamycin B antibiotics, chromopeptide lactones, which contain 3-hydroxypicolinic acid (3-HPA) as the chromophoric group. The enzyme catalyzes both the 3-HPA-dependent ATP-pyrophosphate exchange and the formation of 3-HPA adenylate from 3-HPA and ATP. SDS-polyacrylamide gel electrophoresis indicates that the enzyme is a single polypeptide chain with a Mr between 56,000 and 58,000. The molecular mass of the native enzyme was in the same range. In addition to 3-HPA, the enzyme catalyzes the formation of adenylates from picolinic acid, nicotinic acid, and 2-pyrazinecarboxylic acid. Nicotinic acid and picolinic acid when added externally to etamycin producing S. griseoviridus cultures gave rise to the formation of etamycin analogues each containing nicotinic acid or picolinic acid instead of the genuine 3-HPA. The data strongly suggest that the enzyme is involved in the biosynthesis of the chromopeptide lactone etamycin and possibly in that of other mikamycin B antibiotics.
Neoviridogrisein II is a homologue of viridogrisein in which the hydroxyproline residue is replaced by proline. Neoviridogrisein II proved to be more active than the parent antibiotic against Gram-positive bacteria and Mycoplasma species. When neoviridogrisein II or viridogrisein was combined with griseoviridin, a non-peptidyl macrocyclic lactone, synergism was observed: maximum synergistic effect was observed for a combination ratio that depended on the test bacterium used. Neoviridogrisein II also exerted synergism when combined with mikamycin A and A-2315A.
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