Kiyoshi Kurahashi

Biosynthesis of tyrocidine by a cell-free enzyme system of Bacillus brevis ATCC 8185: I. Preparation of partially purified enzyme system and its properties☆

Abstract 1. A tyrocidine-synthesizing system was partially purified from the crude extract of Bacillus brevis by ammonium sulfate fractionation, protamine sulfate treatment followed by another ammonium sulfate fractionation to a specific activity of about 20 mμmoles of tyrocidine formation per h per mg of protein. The partially purified system absolutely required Mg 2+ (or Mn 2+ ), ATP and the tyrocidine constituent amino acids for its activity. The optimum conditions for tyrocidine synthesis were found to be 2 mM Mg 2+ (or Mn 2+ ), 4 mM ATP, and pH 7.8 in the presence of 0.2–0.4 mM constituent amino acids. 2. AMP was formed from ATP in the process of tyrocidine synthesis and 1 molecule of ATP was required for the formation of one peptide bond. 3. The partially purified system catalyzed ATP-PP i exchange reactions in the presence of glycine, l -alanine, l -lysine and l -isoleucine, besides 9 tyrocidine constituent l -amino acids. The other non-constituent amino acids of tyrocidine did not promote the exchange reaction. 4. Column chromatography on DEAE-cellulose separated the partially purified system into two distinct components, Components I and II, both of which were required for tyrocidine synthesis.

Peptide antibiotic subtilin is synthesized via precursor proteins

Biogenesis of subtilin, an antimicrobial peptide produced by Bacillus subtilis ATCC 6633, was studied in growing cells. Pulse-chase labeling experiments with [35S]cysteine revealed the presence of precursor proteins of subtilin. The synthesis of both precursor proteins and subtilin was inhibited by inhibitors of protein and RNA synthesis. When the precursor proteins were incubated with crude extracts of the organism in vitro, they were converted to subtilin. Pepstatin and phenylmethylsulfonyl fluoride in combination inhibited this conversion.

Biosynthesis of tyrocidine by a cell-free enzyme system of Bacillus brevis ATCC 8185. II. Amino acid substitution in tyrocidine.

Abstract 1. 1. The partially purified tyrocidine-synthesizing system of Bacillus brevis ATCC 8185 was revealed to have the ability to synthesize tyrocidines A, B, C and D. 2. 2. The enzyme system is not absolutely specific for particular amino acids. A new tyrocidine, tyrocidine E (tyrosine residue in tyrocidine A was replaced by phenylalanine), lysyl-tyrocidine A (ornithine residue in tyrocidine A was replaced by lysine) and isoleucyl-tyrocidine A (leucine residue in tyrocidine A was replaced by isoleucine) were synthesized in vitro. 3. 3. A single enzyme system catalyzed the synthesis of these tyrocidines and their formation was directly influenced by the amino acid concentrations in the reaction mixture. 4. 4. Some amino acid analogues, such as 5-methyltryptophan, p-fluorotryptophan, thienylalanine and p-fluorophenylalanine promoted the ATP-32PP1 exchange reaction and were incorporated into tyrocidine.

Mutations in Escherichia coli that affect uridine diphosphate glucose pyrophosphorylase activity and galactose fermentation

Abstract The metabolism of galactose was studied in two galactose-non-fermenting mutants which were isolated from Escherichia coli strain K12 and classified genetically as Group E by Drs. J. and E. M. Lederberg . One of them, W4597, was found to have a single defect in UDPG pyrophosphorylase (UTP: α- d -glucose-1-phosphate uridylyltranserase, EC 2.7.7.9) and to be normal in galactokinase (ATP: d -galactose 1-phosphotransferase, EC 2.7.1.6), Gal-1- P uridyl transferase (UDP glucose: α- d -galactose-1-phosphate uridylyl transferase, EC 2.7.7.12), UDP galactose 4-epimerase (UDP glucose 4-epimerase, EC 5.1.3.2), phosphoglucomutase ( d -glucose-1,6-diphosphate: d -glucose-1-phosphate phosphotransferase, EC 2.7.5.1), and also in galactose permease. Non-fermentation of galactose in this strain was, therefore, ascribed to the defect of UDPG pyrophosphorylase. The other strain, W3142, was shown to have a secondary defect in galactokinase in addition to a defect in UDPG pyrophosphorylase. When grown in the presence of galactose, W4597 accumulated intracellularly a large amount of Gal-1- P . The intracellular levels of UDPG and uridine diphosphate galactose were shown to be much lower in both of these strains than in wild-type strains, irrespective of the presence or absence of galactose in the growth media. The sugars of their cell walls were composed only of an aldoheptose, hexosamines and a smaller amount of glucose; any traces of galactose, which is present in wild type cell walls, were not detected in their cell walls.

Biosynthesis of polymyxin E III. Total synthesis of polymyxin E by a cell-free enzyme system

Abstract Polymyxin E, an antimicrobial branched cyclic decapeptide, was synthesized by an enzyme fraction partially purified from crude extracts of the producing organism, Aerobacillus polyaerogenes . For the synthesis, three constituent amino acids (L-2,4-diaminobutyric acid, L-leucine, and L-threonine), ATP, Mg2+ and an acylating system consisting of octanoyl CoA and an ammonium sulfate fraction of cell extracts are required.

Formylation of enzyme-bound valine and stepwise elongation of intermediate peptides of gramicidin A by a cell-free enzyme system

Summary A partially purified enzyme fraction (Component I) of crude extracts of Bacillus brevis ATCC 8185 which produces gramicidin A activated L-valine and bound it as a thioester, and further formylated it in the presence of a formyltetrahydrofolate synthesizing system. The same fraction catalyzed the synthesis of enzyme-bound formylvalylglycine. The addition of Component II, which was also partially purified, together with the constituent amino acids of gramicidin A brought about the synthesis of enzyme-bound formylvalylglycylalanine and formylvalylglycylalanylleucine (or a longer intermediate).

Identification of the sporulation gene spoOA product of Bacillus subtilis

A 2.4-kilobase fragment of the Bacillus subtilis chromosome containing the wild-type spoOA gene derived from the phi 105dspoOA+-Bc-1 transducing phage was cloned onto plasmid pBR322 in Escherichia coli. A recombinant plasmid harboring the mutant spoOA12 allele on the 2.4-kilobase insert was also constructed from the phi 105dspoOA12-1 phage DNA and pBR322. Protein products synthesized in response to plasmid DNA in a DNA-directed cell-free system derived from E. coli were analyzed by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. A protein of approximately 27,500 daltons synthesized with the recombinant plasmid DNA harboring the wild-type spoOA gene as template was not formed with the recombinant plasmid DNA harboring the spoOA12 allele. Since the spoOA12 mutation is a nonsense mutation, we conclude that the 27.5-kilodalton protein is the product of the spoOA gene.

Two Mechanisms of Biosyntheses of Antibiotic Peptides

The biosynthesis of antibiotic peptides such as gramicidin S, tyrocidines, bacitracins, gramicidin A, and polymyxin E, is carried out by the multienzyme thiotemplate mechanism without the involvement of either ribosomes or mRNAs. However, a larger antibiotic peptide produced by Bacillus subtilis ATCC 6633, subtilin, with 32 amino acid residues, was found to be formed by processing of its precursor protein. There are two different mechanisms for the synthesis of antibiotic oligopeptides by microorganisms.