The Linear Pentadecapeptide Gramicidin Is Assembled by Four Multimodular Nonribosomal Peptide Synthetases That Comprise 16 Modules with 56 Catalytic Domains

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 ␤-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-pentadecapeptideethanolamine. However, initial biochemical analysis of this reductase showed only a one-step reduction yielding the corresponding aldehyde in vitro.Gramicidin is a pentadecapeptide antibiotic produced by Bacillus brevis ATCC 8185 during its sporulation phase (1). The primary structure of gramicidin A was determined as formyl-

Structure and Biosynthesis of the BT Peptide Antibiotic from Brevibacillus texasporus

“…A similar reductase domain has also been identified in the gramicidin A NRPS (9). All three reductases have been experimentally demonstrated to reduce their substrates to corresponding aldehydes in an NADPH-dependent reaction (5,9,19). For myxochelin A and gramicidin A, the aldehydes are further reduced to alcohols.…”

“…Thioesterase-mediated release of the mature peptide from the NRPS enzyme involves transient formation of an acyl-O-TE intermediate that is then hydrolyzed or hydrolyzed and concomitantly cyclized to release the mature peptide (7). An alternative termination scheme involves reduction of the tethered C-terminal residue by a reductase domain (R-domain) that resides in the last NRPS module, resulting in release of a peptide with an alcoholic C-terminal residue (5,9). Such a reductase-mediated termination/C-terminal modification occurs in BT biosynthesis and contributes to superprotease resistance of the BT peptides.…”

Biosynthesis of Fusarubins Accounts for Pigmentation of Fusarium fujikuroi Perithecia

“…In contrast to TE and TE/CLC domains, R domains show sequence similarities to the short-chain dehydrogenase/reductase (SDR) superfamily, exhibiting Rossman fold structure and nucleotide binding motifs (36). ClustalW alignment of the Fsr1 R domain with characterized R domains from bacterial and fungal PKSs (5,13,38,45) and the R domain of the yeast ␣-aminoadipate reductase Lys2p (25) shows high amino acid conservation (see Fig. S2 in the supplemental material).…”

Chemoenzymatic and Template-Directed Synthesis of Bioactive Macrocyclic Peptides

“…7B) (140). Moreover, peptide release can occur under reduction of the carboxy group mediated by the NAD(P)H-dependent reduction domain (R-domain) such as in the biosynthesis of the linear peptide alcohol gramicidin A in B. brevis (62) and in the formation of the macrocyclic imine nostocyclopeptide 8 ( Fig. 1) from Nostoc sp.…”