Journal Article Mechanism of Action of Tetrocarcin A Get access Tatsuya Tamaok, Tatsuya Tamaok Tokyo Research Laboratory, Kyowa Hakko Kogyo Co. Ltd, Machida Tokyo 194, Japan Search for other works by this author on: Oxford Academic Google Scholar Fusao Tomita, Fusao Tomita Tokyo Research Laboratory, Kyowa Hakko Kogyo Co. Ltd, Machida Tokyo 194, Japan Search for other works by this author on: Oxford Academic Google Scholar Yukiteru Obi, Yukiteru Obi Tokyo Research Laboratory, Kyowa Hakko Kogyo Co. Ltd, Machida Tokyo 194, Japan Search for other works by this author on: Oxford Academic Google Scholar Fujio Kawamura, Fujio Kawamura Institute of Applied Microbiology, The University of Tokyo, Bunkyo-ku Tokyo 113, Japan Search for other works by this author on: Oxford Academic Google Scholar Hiuga Saito Hiuga Saito Institute of Applied Microbiology, The University of Tokyo, Bunkyo-ku Tokyo 113, Japan Search for other works by this author on: Oxford Academic Google Scholar Agricultural and Biological Chemistry, Volume 47, Issue 1, 1 January 1983, Pages 59–65, https://doi.org/10.1080/00021369.1983.10865585 Published: 01 January 1983 Article history Received: 20 July 1982 Published: 01 January 1983
Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a beta-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60 degrees C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50 degrees C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only beta-2,6-linkage of levan, but also beta-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-beta-D-fructan 6-levanbiohydrolase (EC 3.2.1.64).
Identification of p-aminophenylalanine (PAP) was made in the culture broth of Corynebacterium hydrocarboclastus and its intracellular pool decreased as progress of Corynecin production. The specific inhibitor of Corynecin biosynthesis, p-aminophenylethylalcohol, caused the accumulation of PAP as well as other aromatic amines related to Corynecins. Variation of PAP pool was consistent with properties as the precursor of Corynecin. Addition of PAP to the medium did not increase the amount of Corynecin production, suggesting that the biosynthesis of Corynecins might be regulated at stages after the formation of PAP. Growth inhibition by PAP added to the medium was observed (5mM for 50% inhibition) and this inhibition was relieved by the addition of tyrosine and phenylalanine but not by shikimate, prephenate or tryptophan, showing that PAP did not inhibit the pathway before the formation of prephanate. These effects of PAP on shikimate pathway seem to be preferable for efficient production of Corynecins.
Tetrocarcin A strongly inhibited incorporation of 14C-uracil and 14C-D-glucosamine into B. subtilis in vivo. It had no effect on PM2 DNA in vitro. It caused 50% inhibition of DNA dependent RNA polymerase from E. coli at a concentration of about 0.1mM, while B. subtilis RNA polymerase was inhibited only 20-30% at a concentration of 0.08 μm and the degree of inhibition did not increase at a higher concentration. The fact that inhibition of RNA synthesis was abolished in permeabilized cells of B. subtilis and leakage of cellular constituents was caused by tetrocarcin Asuggests that RNA synthesis is not the primary target and cell membrane is affected by tetrocarcin A in B. subtilis.
For the purpose of improving the productivity of corynecins (chloramphenicol analogs), the mutation study of the producer, Corynebacterium hydrocarboclastus, was concerned in this investigation with the isolation of glycolipids defective strains. The mutant KY 8834 possessed glycolipids less than one-third to one-tenth of the parent. Productivity of corynecins of the mutant was better than that of the parent. In addition, this character of the mutant was advantageous for the stirring fermentation, because aggregation of cells was remarkably diminished unlike the case of the parental strain. Another mutant was isolated by mutagenesis of glycolipids defective mutant, KY 8834. The mutant, KY 8835, produced dominantly corynecin I (less toxic homolog to the producer strain). Thus, the productivity of corynecins was elevated to about two-fold of that of strain KY 8834.
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