Enzyme-bound formylvaline and formylvalylglycine; an initiation complex for gramicidin A biosynthesis.

Gramicidin A is an antibiotic peptide produced by Bacillus brevis ATCC 8185, which also produces tyrocidines. An attempt was made to establish a cell-free enzyme system for gramicidin A synthesis. An enzyme fraction, Component I, was partially purified from crude extracts of the organism and proven to be involved in the synthesis of the formyl-Val-Gly- region of gramicidin A. The initiation of gramicidin A biosynthesis is a function of Component I, which activates valine and binds it as a thioester, and further formylates it in the presence of formyltetrahydrofolic acid. The formylvaline thus synthesized is transferred to the glycine moiety, which is also thioesterified to Component I. Elongation of the peptide chain takes place by a mechanism similar to those found for tyrocidines, gramicidin S, and bacitracin.     

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

A protein, tentatively named apo-Q-protein I, with molecular weight of 15,000¹ from the reconstitutively active cytochrome $\text{b-c}{}_1$ complex has been identified as being responsible for electron transfer between succinate dehydrogenase and ubiquinone. The identification was based on the chemical modification, proteolytic enzyme digestion, and isolation and purification of the protein to nearly pure form.     

Biosynthesis of gramicidin S with the aid of dipeptides by gramicidin S synthetase.

Dipeptides L-phenylalanyl-proline, D-phenylalanyl-proline, prolyl-valine, valyl-lysine, lysyl-leucine and leucyl-phenylalanine, derived from the sequence of gramicidin S, are substrates of the gramicidin S synthetase. When any of these dipeptides are used to replace the two corresponding amino acids in the reaction assay, cyclodecapeptide antibiotic synthesis occurs, and requires the whole multienzyme system. Active esters, like the thiophenyl and p-nitrophenyl esters of D-phenylalanyl-proline are unable to promote gramicidin S biosynthesis with the gramicidin S synthetase system or with the heavy enzyme alone.     

An enzyme-bound intermediate in the biosynthesis of 3-hydroxy-3-methylglutaryl-coenzyme A

1. Purified 3-hydroxy-3-methylglutaryl-CoA synthase from baker's yeast (free from acetoacetyl-CoA thiolase activity) catalysed an exchange of acetyl moiety between 3'-dephospho-CoA and CoA. The exchange rate was comparable with the overall velocity of synthesis of 3-hydroxy-3-methylglutaryl-CoA. 2. Acetyl-CoA reacted with the synthase, giving a rapid ;burst' release of CoA proportional in amount to the quantity of enzyme present. The ;burst' of CoA was released from acetyl-CoA, propionyl-CoA and succinyl-CoA (3-carboxypropionyl-CoA) but not from acetoacetyl-CoA, hexanoyl-CoA, dl-3-hydroxy-3-methylglutaryl-CoA, or other derivatives of glutaryl-CoA. 3. Incubation of 3-hydroxy-3-methylglutaryl-CoA synthase with [1-(14)C]acetyl-CoA yielded protein-bound acetyl groups. The K(eq.) for the acetylation was 1.2 at pH7.0 and 4 degrees C. Acetyl-labelled synthase was isolated free from [1-(14)C]acetyl-CoA by rapid gel filtration at pH6.1. The [1-(14)C]acetyl group was removed from the protein by treatment with hydroxylamine, CoA or acetoacetyl-CoA but not by acid. When CoA or acetoacetyl-CoA was present the radioactive product was [1-(14)C]acetyl-CoA or 3-hydroxy-3-methyl-[(14)C]glutaryl-CoA respectively. 4. The isolated [1-(14)C]acetyl-enzyme was slowly hydrolysed at pH6.1 and 4 degrees C with a first-order rate constant of 0.005min(-1). This rate could be stimulated either by raising the pH to 7.0 or by the addition of desulpho-CoA. 5. These properties are interpreted in terms of a mechanism in which 3-hydroxy-3-methyl-glutaryl-CoA synthase is acetylated by acetyl-CoA to give a stable acetyl-enzyme, which then condenses with acetoacetyl-CoA yielding a covalent derivative between 3-hydroxy-3-methylglutaryl-CoA and the enzyme which is then rapidly hydrolysed to free enzyme and product.     

Organization of the biosynthesis genes for the peptide antibiotic gramicidin S.

A recombinant bacteriophage containing the intact Bacillus brevis gene for gramicidin S synthetase 1, grsA, and the 5' end of the gramicidin S synthetase 2 gene, grsB, was identified by screening an EMBL3 library with anti-GrsA antibodies. This clone, EMBL315, has a 14-kilobase (kb) insert that hybridizes to the previously isolated 3.9-kb fragment of the grsB gene, which encodes the 155-kilodalton ornithine-activating domain of gramicidin S synthetase 2. Deletion and subcloning experiments with the 14-kb insert located the grsA structural gene and its putative promoter on a 4.5-kb PvuII fragment which encoded the full-length 120-kilodalton protein in Escherichia coli. In addition, hybridization analysis revealed that the 5' end of the grsB gene is located approximately 3 kb from the grsA structural gene. Furthermore, these studies indicated that grsA and grsB are transcribed in opposite orientations.     

The initiation of fetal hemoglobin biosynthesis.

Biosynthesis of the alpha and beta chains of rabbit and human adult hemoglobin is initiated with a methionyl residue, which is removed during elongation of the peptide chain. To study the initiation of biosynthesis of the delta chain of human fetal hemoglobin, fresh placental blood was used for labeling experiments with radioactive amino acids. Labeled nascent peptide chains were purified from the polysomal fraction of placental blood reticulocytes. The number of amino acid residues in nascent gamma chain at the time of removal of its N-terminal methionine was estimated to be 40--60 from the relative yields of labeled tryptic peptides.     

Growth limitations and biosynthesis of gramicidin in Bacillus brevis var. G.-B

Gramicidin S biosynthesis was studied in Bacillus brevis var. G.-B. during its batch and continuous cultivation when the culture growth was limited with nutrient sources (glycerol, ammonium nitrogen, phosphate), oxygen deficiency and the action of a physical factor (a low temperature). The antibiotic biosynthesis was shown to be induced by a change in the growth rate caused by the action of any factor decelerating the growth. The authors propose a mathematical model for the antibiotic synthesis, biomass accumulation and the utilization of a substrate limiting the growth. The model is based on the age separation of cells. The model is analyzed in terms of optimizing the one-stage continuous cultivation process. The model allows one to calculate optimal conditions of the antibiotic synthesis in the process of one-stage continuous cultivation.     

Structure of gramicidin A.

Gramicidin A, a hydrophobic linear polypeptide, forms channels in phospholipid membranes that are specific for monovalent cations. Nuclear Magnetic Resonance (NMR) spectroscopy provided the first direct physical evidence that the channel conformation in membranes is an amino terminal-to-amino terminal helical dimer, and circular dichroism (CD) spectroscopy has shown the sensitivity of its conformation to different environments and the structural consequences of ion binding. The three-dimensional structure of a gramicidin/cesium complex has been determined by x-ray diffraction of single crystals using single wavelength anomalous scattering for phasing. The left-handed double helix in this crystal form corresponds to one of the intermediates in the process of folding and insertion into membranes. Co-crystals of gramicidin and lipid that appear to have gramicidin in their membrane channel conformation have also been formed and are presently under investigation. Hence, we have used a combination of spectroscopic and diffraction techniques to examine the conformation and functionally-related structural features of gramicidin A.     

Purification of gramicidin A.

A simple chromatographic purification of the naturally occurring ion channel-forming pentadecapeptide gramicidin A (gA) is presented. This procedure allows gA to be isolated in gram quantities from the commercially available mixture of isomers after chromatography on silica gel. The gramicidin A obtained in this manner is greater than 95% pure as determined by 1HNMR, HPLC, and amino acid analysis.     

Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S.

The non-ribosomal synthesis of the cyclic peptide antibiotic gramicidin S is accomplished by two large multifunctional enzymes, the peptide synthetases 1 and 2. The enzyme complex contains five conserved subunits of approximately 60 kDa which carry out ATP-dependent activation of specific amino acids and share extensive regions of sequence similarity with adenylating enzymes such as firefly luciferases and acyl-CoA ligases. We have determined the crystal structure of the N-terminal adenylation subunit in a complex with AMP and L-phenylalanine to 1.9 A resolution. The 556 amino acid residue fragment is folded into two domains with the active site situated at their interface. Each domain of the enzyme has a similar topology to the corresponding domain of unliganded firefly luciferase, but a remarkable relative domain rotation of 94 degrees occurs. This conformation places the absolutely conserved Lys517 in a position to form electrostatic interactions with both ligands. The AMP is bound with the phosphate moiety interacting with Lys517 and the hydroxyl groups of the ribose forming hydrogen bonds with Asp413. The phenylalanine substrate binds in a hydrophobic pocket with the carboxylate group interacting with Lys517 and the alpha-amino group with Asp235. The structure reveals the role of the invariant residues within the superfamily of adenylate-forming enzymes and indicates a conserved mechanism of nucleotide binding and substrate activation.