anticapsin and bacilysin

Biosynthesis of the dipeptide antibiotic bacilysin, encoded by the seven Bacillus subtilis genes bacA-G, involves diversion of flux from prephenate to the noncognate amino acid anticapsin. The anticapsin warhead is then ligated to the C-terminus of l-alanine to produce mature bacilysin. We have previously noted the formation of two diastereomers of tetrahydrotyrosine (4S- and 4R-H(4)Tyr) by tandem action of the four purified enzymes BacABGF. BacC (oxidase) and BacD (ligase) have been hypothesized to be remaining late stage enzymes in bacilysin biosynthesis. Using a combination of BacCD in vitro studies, B. subtilis deletion mutants, and isotopic feeding studies, we were able to determine that the H(4)Tyr diastereomers are actually shunt products that are not on-pathway to bacilysin biosynthesis. Dihydroanticapsin and dihydrobacilysin accumulate in extracts of a ΔbacC strain and are processed to anticapsin and then bacilysin upon addition of BacC and BacD, respectively. These results suggest the epoxide group in bacilysin is installed in an earlier step of bacilysin biosynthesis, while BacC oxidation of the C(7)-hydroxyl and the subsequent BacD ligation of anticapsin to l-Ala are the penultimate and ultimate steps of bacilysin biosynthesis, respectively.

Topics: Alanine; Anti-Bacterial Agents; Bacillus subtilis; Dipeptides; Gene Deletion; Ligases; Oxidoreductases; Tyrosine

In Bacillus subtilis, bacilysin is a nonribosomally synthesized dipeptide antibiotic composed of L-alanine and L-anticapsin. The biosynthesis of bacilysin depends on the bacABCDEywfG operon (bac operon)and the adjacent ywfH gene. To elucidate the effects of global regulatory genes on the expression of bac operon, we used the combination of lacZ fusion analysis and the gel mobility shift assays. The cell density-dependent transition state induction of the bac operon was clearly shown. The basal expression level of the bac operon as well as transition state induction of bac is directly ComA dependent. Three Phr peptides, PhrC, PhrF and PhrK, are required for full-level expression of ComA-dependent bac operon expression, but the most important role seemed to be played by PhrC in stimulating bac expression through a RapC-independent manner. Spo0A is another positive regulator which participates in the transition state induction of bac both directly by interacting with the bac promoter and indirectly by repressing abrB expression. AbrB and CodY proteins do not only directly repress the bac promoter, but they also mutually stimulate the transition state induction of bac indirectly, most likely by antagonizing their repressive effects without preventing each other's binding since both proteins can bind to the bac promoter simultaneously.

Topics: Alanine; Anti-Bacterial Agents; Bacillus subtilis; Bacterial Proteins; Bacteriocins; Dipeptides; DNA-Binding Proteins; DNA, Bacterial; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Bacterial; Genes, Bacterial; Lac Operon; Mutation; Promoter Regions, Genetic; Quorum Sensing; Repressor Proteins; Transcription Factors

Bacillus subtilis produces the antibiotic anticapsin as an L-Ala-L-anticapsin dipeptide precursor known as bacilysin, whose synthesis is encoded by the bacA-D genes and the adjacent ywfGH genes. To evaluate the biosynthesis of the epoxycyclohexanone amino acid anticapsin from the primary metabolite prephenate, we have overproduced, purified, and characterized the activity of the BacA, BacB, YwfH, and YwfG proteins. BacA is an unusual prephenate decarboxylase that avoids the typical aromatization of the cyclohexadienol ring by protonating C(8) to produce an isomerized structure. BacB then catalyzes an allylic isomerization, generating a conjugated dienone with a 295 nm chromophore. Both the BacA and BacB products are regioisomers of H(2)HPP (dihydro-4-hydroxyphenylpyruvate). The BacB product is then a substrate for the short chain reductase YwfH which catalyzes the conjugate addition of hydride at the C(4) olefinic terminus using NADH to yield the cyclohexenol-containing tetrahydro-4-hydroxyphenylpyruvate H(4)HPP. In turn, this keto acid is a substrate for YwfG, which promotes transamination (with L-Phe as amino donor), to form tetrahydrotyrosine (H(4)Tyr). Thus BacA, BacB, YwfH, and YwfG act in sequence in a four enzyme pathway to make H(4)Tyr, which has not previously been identified in B. subtilis but is a recognized building block in cyanobacterial nonribosomal peptides such as micropeptins and aeruginopeptins.

Topics: Alanine; Amino Acids, Aromatic; Amino Acids, Dicarboxylic; Bacillus subtilis; Bacterial Proteins; Carbon-Carbon Double Bond Isomerases; Carboxy-Lyases; Cyclohexanecarboxylic Acids; Cyclohexenes; Dipeptides; Oxidoreductases Acting on CH-CH Group Donors; Prephenate Dehydratase; Signal Transduction; Transaminases; Tyrosine

Bacilysin is a non-ribosomally synthesized dipeptide antibiotic that is active against a wide range of bacteria and some fungi. Synthesis of bacilysin (l-alanine-[2,3-epoxycyclohexano-4]-l-alanine) is achieved by proteins in the bac operon, also referred to as the bacABCDE (ywfBCDEF) gene cluster in B. subtilis. Extensive genetic analysis from several strains of B. subtilis suggests that the bacABC gene cluster encodes all the proteins that synthesize the epoxyhexanone ring of l-anticapsin. These data, however, were not consistent with the putative functional annotation for these proteins whereby BacA, a prephenate dehydratase along with a potential isomerase/guanylyl transferase, BacB and an oxidoreductase, BacC, could synthesize l-anticapsin. Here we demonstrate that BacA is a decarboxylase that acts on prephenate. Further, based on the biochemical characterization and the crystal structure of BacB, we show that BacB is an oxidase that catalyzes the synthesis of 2-oxo-3-(4-oxocyclohexa-2,5-dienyl)propanoic acid, a precursor to l-anticapsin. This protein is a bi-cupin, with two putative active sites each containing a bound metal ion. Additional electron density at the active site of the C-terminal domain of BacB could be interpreted as a bound phenylpyruvic acid. A significant decrease in the catalytic activity of a point variant of BacB with a mutation at the N-terminal domain suggests that the N-terminal cupin domain is involved in catalysis.

Topics: Alanine; Bacillus subtilis; Bacterial Proteins; Catalysis; Crystallography, X-Ray; Dipeptides; Magnetic Resonance Spectroscopy; Mutation; Protein Conformation

The genes encoding the biosynthesis of the dipeptide bacilysin and its antibiotic constituent anticapsin were isolated from several strains of Bacillus subtilis as well as B. amyloliquefaciens and B. pumilus. The ywfBCDEF genes of B. subtilis 168 were shown to carry the biosynthetic core functions and were renamed bacABCDE. Mutation of the bacD gene or transformation of the bacABC genes into a B. subtilis Delta (ywfA-bacABCDE) deletion mutant led to the accumulation of anticapsin, which was fourfold higher after transformation of the bacABC genes into a bacD mutant. The genes bacD and bacE proved to encode the functions of amino acid ligation and self-protection to bacilysin, respectively. Amplification of the bacABCDE gene cluster in a bacAB gene-deficient host strain of B. amyloliquefaciens resulted in a tenfold bacilysin overproduction. Some host strains required distinct glucosamine and yeast extract supplements in order to prevent suicidal effects of the recombinant antibiotic production. The bac genes from different Bacillus species revealed the same arrangement and 72.6-88.6% of sequence identity.

Topics: Alanine; Bacillus; Dipeptides; Genes, Bacterial; Recombinant Proteins

A dipeptide antibiotic, tetaine, was found to diminish the rate of incorporation of 3H-labelled precursors into nucleic acids of intact and permeabilized HeLa S3 cells with concomitant negligible effect on protein synthesis. Comparison of the inhibitory effects of tetaine indicates that the antibiotic at 0.03-0.1 mM is a selective inhibitor of cellular DNA biosynthesis and, at higher concentration, of DNA and RNA biosynthesis. Tetaine is also an inhibitor of DNA and RNA polymerase reactions in a cell-free system, as determined using partially purified extracts from HeLa S3 cells that served as a source of the enzymes. The pretreatment experiments showed that tetaine inactivated the polymerases without affecting DNA template function. The tetaine effect on biosynthesis of nucleic acids in HeLa S3 cells can be attributed rather to the intact antibiotic than to the product of its enzymatic cleavage, anticapsin.

Topics: Alanine; Dipeptides; DNA Replication; DNA-Directed RNA Polymerases; HeLa Cells; Humans; Nucleic Acid Synthesis Inhibitors; Protein Biosynthesis; RNA; Templates, Genetic

L-Glutamine:D-fructose-6-phosphate amidotransferase ('glucosamine synthase', EC 5.3.1.19) from Escherichia coli MRE 600 was purified at least 75-fold. It catalysed the formation of 21.1 mumol glucosamine 6-phosphate (mg protein)-1 in 30 min at 37 degrees C. Its molecular weight, estimated by gel filtration, was about 90000 and it was inhibited by thiol group reagents. Anticapsin, the C-terminal amino acid of the dipeptide antibiotic bacilysin, and to a lesser extent bacilysin itself, inhibited glucosamine synthase activity. Kinetic studies indicated that the inhibition was non-competitive with respect to fructose 6-phosphate as substrate but partly competitive with respect to L-glutamine. Incubation of the enzyme with anticapsin brought about a time-dependent and irreversible inhibition. It is suggested that anticapsin behaves as a glutamine analogue and that a reaction of its epoxide group with a thiol group of glucosamine synthase results in its linkage to the enzyme by a covalent bond.

Topics: Alanine; Anti-Bacterial Agents; Carbohydrate Epimerases; Dipeptides; Escherichia coli; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); Kinetics

The dipeptide antibiotic bacilysin is active against a wide range of bacteria and against Candida albicans. Its C-terminal amino acid, anticapsin, is a very poor antibacterial agent. The activities of both substances are strongly dependent on the nature of the culture medium. In a minimal medium the minimum inhibitory concentration for bacilysin with E. coli B is 10(-3) mug ml(-1). The action of bacilysin amino acids. With several bacteria, bacilysin-resistant mutants are found in unusually large numbers. It is suggested that peptide and amino acid transport systems play a role in these phenomena. The antimicrobial action of bacilysin is also inhibited by glucosamine and N-acetylglucosamine. This antibiotic may therefore interfere with glucosamine synthesis and thus with the synthesis of microbial cell walls.

Topics: Acetylglucosamine; Alanine; Anti-Bacterial Agents; Bacillus subtilis; Candida albicans; Chemical Phenomena; Chemistry; Cyclohexanones; Dipeptides; Drug Resistance, Microbial; Escherichia coli; Glucosamine; Mutation; Salmonella typhi; Staphylococcus aureus