Isolation of bacilysin and a new amino acid from culture filtrates of Bacillus subtilis.
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ABSTRACT:
SUBMITTER: Walker JE
PROVIDER: S-EPMC1179251 | biostudies-other | 1970 Jul
REPOSITORIES: biostudies-other
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Project description: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.
| S-EPMC2797259 | biostudies-literature
Project description:Bacillus subtilis mutants with high expression of the bacilysin operon ywfBCDEFG were isolated. Comparative genome sequencing analysis revealed that all of these mutants have a mutation in the scoC gene. The disruption of scoC by genetic engineering also resulted in increased expression of ywfBCDEFG. Primer extension and gel mobility shift analyses showed that the ScoC protein binds directly to the promoter region of ywfBCDEFG. Our results indicate that the transition state regulator ScoC, together with CodY and AbrB, negatively regulates bacilysin production in B. subtilis.
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Project description:RizA is an L-amino-acid ligase from Bacillus subtilis that participates in the biosynthesis of rhizocticin, an oligopeptide antibiotic. The substrate-free form of RizA has been crystallized and the structure was solved at 2.8 Å resolution. The amino-acid-binding site appears to be capable of accommodating multiple amino acids, consistent with previous biochemical studies.
| S-EPMC4555917 | biostudies-literature
Project description:1. Mass spectra of the trimethylsilyl derivative and the methyl ester of the N-trifluoroacetyl derivative of bacilysin indicated that the antibiotic had a molecular weight of 270. Several peaks in the spectrum of the methyl ester were consistent with the presence of an N-terminal alanine residue in the molecule. 2. The proton-magnetic-resonance spectrum of bacilysin confirmed that the antibiotic contained an epoxide group and the spin-spin splitting of the protons of the epoxide group indicated that the side chain of the epoxycyclohexanone ring was attached at C-4 and was alphabeta to the keto group. 3. The formation of an alphabeta-unsaturated ketone on reduction of bacilysin with chromous chloride also showed that the epoxide was alphabeta to the keto group. 4. The optical-rotatory-dispersion curve of bacilysin showed a positive Cotton effect. On the assumption that the reversed Octant rule for alphabeta-epoxyketones was applicable this revealed the absolute stereochemistry and enabled a definitive structure to be assigned to the molecule. 5. Similar measurements showed that substance AA1, isolated from culture supernatants, was the C-terminal amino acid of bacilysin. 6. Hydrolysis of substance P2 with leucine aminopeptidase and the mass spectrum of the methyl ester of its N-trifluoroacetyl derivative showed that this substance was l-analyl-l-alanine. 7. These results are discussed in relation to the biogenesis of bacilysin.
| S-EPMC1179252 | biostudies-other
Project description:In this study, we have identified a bacterium that can inhibit the growth of Staphylococcus aureus, and further analyzed its antibacterial activity and other biological characteristics and laid the foundation for its future application. Through isolation and culture of the unknown bacteria, the culture characteristics, morphology observation, biochemical test, preliminary antibacterial test, 16S rRNA PCR amplification, sequence analysis, and homology analysis were performed. It was found that the bacteria are Gram positive spore chain Bacillus. The bacteria could only ferment glucose for acid production, but could not utilize lactose and maltose. The VP test for this bacteria was positive, while indole and methyl red tests were negative. Further analysis showed that these bacteria shared a homology up to 99.4% with Bacillus subtilis DQ198162.1. Thus, this newly identified bacterium was classified as Bacillus subtilis. Importantly, the crude bacteriocin of this Bacillus subtilis could inhibit the growth of Staphylococcus aureus, Escherichia coli, Enterococcus and Salmonella, which implies its potential usage in the future.
| S-EPMC5880821 | biostudies-literature
Project description:BacD is an ATP-dependent dipeptide ligase responsible for the biosynthesis of L-alanyl-L-anticapsin, a precursor of an antibiotic produced by Bacillus spp. In contrast to the well-studied and phylogenetically related D-alanine: D-alanine ligase (Ddl), BacD synthesizes dipeptides using L-amino acids as substrates and has a low substrate specificity in vitro. The enzyme is of great interest because of its potential application in industrial protein engineering for the environmentally friendly biological production of useful peptide compounds, such as physiologically active peptides, artificial sweeteners and antibiotics, but the determinants of its substrate specificity and its catalytic mechanism have not yet been established due to a lack of structural information. In this study, we report the crystal structure of BacD in complex with ADP and an intermediate analog, phosphorylated phosphinate L-alanyl-L-phenylalanine, refined to 2.5-Å resolution. The complex structure reveals that ADP and two magnesium ions bind in a manner similar to that of Ddl. However, the dipeptide orientation is reversed, and, concomitantly, the entrance to the amino acid binding cavity differs in position. Enzymatic characterization of two mutants, Y265F and S185A, demonstrates that these conserved residues are not catalytic residues at least in the reaction where L-phenylalanine is used as a substrate. On the basis of the biochemical and the structural data, we propose a reaction scheme and a catalytic mechanism for BacD.
| S-EPMC3403468 | biostudies-literature
Project description:Biosynthesis of an Escherichia coli cell, with organic compounds as sources of energy and carbon, requires approximately 20 to 60 billion high-energy phosphate bonds [Stouthamer, A. H. (1973) Antonie van Leeuwenhoek 39, 545-565]. A substantial fraction of this energy budget is devoted to biosynthesis of amino acids, the building blocks of proteins. The fueling reactions of central metabolism provide precursor metabolites for synthesis of the 20 amino acids incorporated into proteins. Thus, synthesis of an amino acid entails a dual cost: energy is lost by diverting chemical intermediates from fueling reactions and additional energy is required to convert precursor metabolites to amino acids. Among amino acids, costs of synthesis vary from 12 to 74 high-energy phosphate bonds per molecule. The energetic advantage to encoding a less costly amino acid in a highly expressed gene can be greater than 0.025% of the total energy budget. Here, we provide evidence that amino acid composition in the proteomes of E. coli and Bacillus subtilis reflects the action of natural selection to enhance metabolic efficiency. We employ synonymous codon usage bias as a measure of translation rates and show increases in the abundance of less energetically costly amino acids in highly expressed proteins.
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Project description:The bioconversion of cellulose and hemicellulose to soluble sugars is important for global stabilization and a sustainable human society. Here, hundreds of cellulolytic bacteria were screened and isolated from soil, compost, and animal waste slurry in Jeju Island, South Korea. Among the isolates, three strains, SL9-9, C5-16, and S52-2, showing higher potential for practical uses were purified on carboxymethyl cellulose (CMC) agar plates and identified as Bacillus subtilis strains by morphological, physiological, and biochemical characterization and 16S rRNA gene analysis. The production patterns of cellulose or hemicellulose-degrading enzymes were investigated during cell culture. All three isolated strains produced CMCase, Avicelase, β-glucosidase, and xylanase enzymes, which suggested synergic cellulolytic systems in Bacillus subtilis. The enzymes showing CMCase, Avicelase, and xylanase activities existed in cell-free culture supernatant, meanwhile β-glucosidase activity was detected in cell debris suggesting that three of the enzymes, including CMCase, Avicelase, and xylanase, were extracellular, and β-glucosidase was cell membrane bound. The three isolates, SL9-9, C5-16, and S52-2, were not the same strains, presenting slight differences in biochemical characteristics, 16S rRNA gene sequences, and cellulolytic enzyme activities.
| S-EPMC3658498 | biostudies-literature
Project description:CodY is a branched-chain amino acid (BCAA) and GTP sensor and a global regulator of transcription in low G + C Gram-positive bacteria. It controls the expression of over 100 genes and operons, principally by repressing during growth genes whose products are required for adaptations to nutrient limitation. However, the mechanism by which BCAA binding regulates transcriptional changes is not clear. It is known that CodY consists of a GAF (cGMP-stimulated phosphodiesterases, adenylate cyclases, FhlA) domain that binds BCAAs and a winged helix-turn-helix (wHTH) domain that binds to DNA, but the way in which these domains interact and the structural basis of the BCAA dependence of this interaction are unknown. To gain new insights, we determined the crystal structure of unliganded CodY from Bacillus subtilis revealing a 10-turn ?-helix linking otherwise discrete GAF and wHTH domains. The structure of CodY in complex with isoleucine revealed a reorganized GAF domain. In both complexes CodY was tetrameric. Size exclusion chromatography with multiangle laser light scattering (SEC-MALLS) experiments showed that CodY is a dimer at concentrations found in bacterial cells. Comparison of structures of dimers of unliganded CodY and CodY-Ile derived from the tetramers showed a splaying of the wHTH domains when Ile was bound; splaying is likely to account for the increased affinity of Ile-bound CodY for DNA. Electrophoretic mobility shift and SEC-MALLS analyses of CodY binding to 19-36-bp operator fragments are consistent with isoleucine-dependent binding of two CodY dimers per duplex. The implications of these observations for effector control of CodY activity are discussed.
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Project description:Bacillus subtilis YwfE, an L-amino-acid ligase, catalyzes the formation of an α-dipeptide from L-amino acids in an ATP-dependent manner. In order to elucidate the substrate-recognition mode and the reaction mechanism of this ligase, native and selenomethionine-derivatized (SeMet) crystals of YwfE in the presence of ADP, MgCl(2) and the dipeptide L-Ala-L-Gln were obtained using the hanging-drop vapour-diffusion method. These crystals diffracted to 1.9 and 2.8 Å resolution, respectively. Preliminary SAD phase calculations using the data set from the SeMet crystal suggested that the crystal belonged to the hexagonal space group P6(5)22, with unit-cell parameters a = b = 90.85, c = 250.31 Å, and contained one molecule in the asymmetric unit with a solvent content of 57.3%.
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