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

Abstract

tase genes have revealed a conserved and ordered modular 4 Corresponding author organization. Each module encodes a functional building unit containing~1000 amino acids, which specifically The non-ribosomal synthesis of the cyclic peptide recognizes a single amino acid. Within such a protein antibiotic gramicidin S is accomplished by two large template-directed peptide biosynthesis, the occurrence and multifunctional enzymes, the peptide synthetases 1 and specific order of the modules in the genomic DNA dictate 2. The enzyme complex contains five conserved subunits the number and sequence of the amino acids to be of~60 kDa which carry out ATP-dependent activation incorporated into the resulting oligopeptide. The modular of specific amino acids and share extensive regions of arrangement of peptide synthetases closely parallels the sequence similarity with adenylating enzymes such multienzyme complexes responsible for the biogenesis of as firefly luciferases and acyl-CoA ligases. We have fatty acids and of the polyketide family of natural products. determined the crystal structure of the N-terminal Furthermore, peptide synthetases, fatty-acid synthetases adenylation subunit in a complex with AMP and and polyketide synthetases all use enzyme-bound phospho-L-phenylalanine to 1.9 Å resolution. The 556 amino pantetheine cofactors as acyl carriers, in a thiotemplate acid residue fragment is folded into two domains with mechanism first proposed by Lipmann more than 20 the active site situated at their interface. Each domain years ago (Lipmann, 1971) and revised recently (Stein of the enzyme has a similar topology to the correspond et al., 1996). ing domain of unliganded firefly luciferase, but aIn particular, the synthesis of the cyclic antibiotic remarkable relative domain rotation of 94°occurs.gramicidin S has been studied in detail. Gramicidin S is This conformation places the absolutely conserved produced by the Gram-positive bacterium Bacillus brevis Lys517 in a position to form electrostatic interactions and consists of two identical pentapeptides joined head with both ligands. The AMP is bound with the phosto tail. It is synthesized by the multienzyme complex phate moiety interacting with Lys517 and the hydroxyl gramicidin S synthetase, which is encoded by the 19 kb groups of the ribose forming hydrogen bonds with grs operon that includes the genes grsA, grsB and grsT. Asp413. The phenylalanine substrate binds in a hydro-The grsT gene, which is located at the 5Ј-end of the grs phobic pocket with the carboxylate group interacting operon, encodes a 29 kDa protein homologous to fattywith Lys517 and the α-amino group with Asp235. The acid thioesterases. The grsA gene product, gramicidin S structure reveals the role of the invariant residues synthetase 1 (GrsA) is a protein composed of 1098 amino within the superfamily of adenylate-forming enzymes acids (Hori et al., 1989; Krätzschmar et al., 1989). and indicates a conserved mechanism of nucleotide GrsA activates L-phenylalanine to the corresponding acylbinding and ...

Use of the Rhodopseudomonas palustris genome sequence to identify a single amino acid that contributes to the activity of a coenzyme A ligase with chlorinated substrates

“…AL3007 have been reported (Gulick et al, 2003;Jogl and Tong, 2004). Crystal structures are also available for three other acyladenylate-forming enzymes; firefly luciferase (Conti et al, 1996), the phenylalanine-activating domain (PheA) of gramicidin S synthetase I from Bacillus brevis (Conti et al, 1997) and the aryl acid-activating domain (2,3-dihydroxy benzoate-AMP ligase, DhbE) of a non-ribosomal peptide synthetase from Bacilius subtilis (May et al, 2002). These structures each reveal an overall protein topology consisting of a large N-terminal domain and a small C-terminal domain.…”

AMP‐forming acetyl‐CoA synthetases in Archaea show unexpected diversity in substrate utilization

“…Although members of this superfamily all catalyze mechanistically similar reactions, they share little identity and similarity in amino acid sequence with the exception of a few signature motifs and conserved core sequence motifs (Babbitt et al 1992, Kleinkauf and Von Dohren 1996, Chang et al 1997. Structures for several members of this family have been determined (Conti et al 1996, Conti et al 1997, May et al 2002, but provide little information regarding the active site and catalytic mechanism of ACS, because they catalyze unrelated reactions in which the intermediates serve different functions and share too little homology to allow structural modeling of ACS.…”

Identification, isolation, and analysis of a gene cluster involved in iron acquisition by Pseudomonas mendocina ymp

“…Each NRPS module incorporates a single substrate into a peptide chain (Ansari et al 2004), where the substrate's identity is encoded by a series of 10 amino acid residues lining the specificity pocket of the module's adenylation domain (http://www-ab.informatik.unituebingen.de/toolbox/) (Conti et al 1997;Rausch et al 2005). Specificity pockets and their corresponding substrates were predicted for each module via sequence comparisons using the NRPS-predictor software.…”