Abstract
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.
Highlights
Gramicidin is a pentadecapeptide antibiotic produced by Bacillus brevis ATCC 8185 during its sporulation phase [1]
We cloned and sequenced a DNA locus of B. brevis ATCC 8185 of about 74 kbp. 61 kbp of this region contain the entire gramicidin biosynthesis operon consisting of four large genes, lgrA, lgrB, lgrC, and lgrD
We found a putative formylation domain upstream of the first module and directly joined to it, seven epimerization domains, and a putative reductase domain attached downstream to module 16
Summary
Three core domains of NRPSs have been identified as being the minimal requirement needed, namely adenylation (A), thiolation (PCP), and condensation (C) domains [5]. The A domain selects the cognate amino acid and activates it as aminoacyl adenylate at the expense of ATP [6, 7]. The activated amino acid is transferred onto the thiol moiety of the downstream PCP domain, giving an energy-rich thioester bond [8]. The C domain is located between two adjacent A-PCP domain pairs It catalyzes the condensation of the thioester-bound intermediates, thereby elongating the peptide chain by 1 amino acid [9]. The chain that is attached to the downstream PCP domain is subsequently used in the condensation reaction of the C domain and by consequent elongation handed on until it reaches the terminal PCP domain of the NRPS. We describe here the cloning and sequencing of the gramicidin biosynthetic gene cluster encoding four large NRPSs and provide biochemical data for the colinearity of this gigantic assembly line
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