gramicidin-a and formic-acid

Linear gramicidin is a membrane channel forming pentadecapeptide that is produced via the nonribosomal pathway. It consists of 15 hydrophobic amino acids with alternating l- and d-configuration forming a beta-helix-like structure. It has an N-formylated valine and a C-terminal ethanolamine. Here we report cloning and sequencing of the entire biosynthetic gene cluster as well as initial biochemical analysis of a new reductase domain. The biosynthetic gene cluster was identified on two nonoverlapping fosmids and a 13-kilobase pair (kbp) interbridge fragment covering a region of 74 kbp. Four very large open reading frames, lgrA, lgrB, lgrC, and lgrD with 6.8, 15.5, 23.3, and 15.3 kbp, were identified and shown to encode nonribosomal peptide synthetases with two, four, six, and four modules, respectively. Within the 16 modules identified, seven epimerization domains in alternating positions were detected as well as a putative formylation domain fused to the first module LgrA and a putative reductase domain attached to the C-terminal module of LgrD. Analysis of the substrate specificity by phylogenetic studies using the residues of the substrate-binding pockets of all 16 adenylation domains revealed a good agreement of the substrate amino acids predicted with the sequence of linear gramicidin. Additional biochemical analysis of the three adenylation domains of modules 1, 2, and 3 confirmed the colinearity of this nonribosomal peptide synthetase assembly line. Module 16 was predicted to activate glycine, which would then, being the C-terminal residue of the peptide chain, be reduced by the adjacent reductase domain to give ethanolamine, thereby releasing the final product N-formyl-pentadecapeptide-ethanolamine. However, initial biochemical analysis of this reductase showed only a one-step reduction yielding the corresponding aldehyde in vitro.

Topics: Adenine; Aldehydes; Amino Acid Sequence; Amino Acids; Bacillus; Bacillus subtilis; Catalytic Domain; Cloning, Molecular; DNA, Bacterial; Ethanolamine; Formates; Glycine; Gramicidin; Molecular Sequence Data; Open Reading Frames; Oxidoreductases; Peptide Synthases; Phylogeny; Protein Structure, Secondary; Sequence Alignment; Sequence Analysis, DNA; Substrate Specificity

The addition of 2 M formic acid at pH 3.75 increased the single channel H+ ion conductance of gramicidin channels 12-fold at 200 mV. Other weak acids (acetic, lactic, oxalic) produce a similar, but smaller increase. Formic acid (and other weak acids) also blocks the K+ conductance at pH 3.75, but not at pH 6.0 when the anion form predominates. This increased H+ conductance and K+ block can be explained by formic acid (HF) binding to the mouth of the gramicidin channel (Km = 1 M) and providing a source of H+ ions. A kinetic model is derived, based on the equilibrium binding of formic acid to the channel mouth, that quantitatively predicts the conductance for different mixtures of H+, K+, and formic acid. The binding of the neutral formic acid to the mouth of the gramicidin channel is directly supported by the observation that a neutral molecule with a similar structure, formamide (and malonamide and acrylamide), blocks the K+ conductance at pH 6.0. The H+ conductance in the presence of formic acid provides a lower bound for the intrinsic conductance of the gramicidin channel when there is no diffusion limitation at the channel mouth. The 12-fold increase in conductance produced by formic acid suggests that greater than 90% of the total resistance to H+ results from diffusion limitation in the bulk solution.

Topics: Amides; Carboxylic Acids; Electric Conductivity; Formates; Gramicidin; Hydrogen-Ion Concentration; Ion Channels; Mathematics; Models, Biological; Potassium; Protons

It is shown that N-formylation of the tryptophan residues of gramicidin completely and reversibly blocks the hexagonal HII phase-inducing ability of the peptide in dioleoylphosphatidylcholine model membranes.

Topics: Amino Acid Sequence; Chemical Phenomena; Chemistry; Formates; Gramicidin; Lipid Bilayers; Liposomes; Magnetic Resonance Spectroscopy; Membrane Lipids; Phosphatidylcholines; Structure-Activity Relationship; Tryptophan

Topics: Bacillus; Cell-Free System; Ethanolamine; Ethanolamines; Formates; Gramicidin; Serine; Valine