Structural Basis for the Activation of Phenylalanine in the Non-Ribosomal Biosynthesis of Gramicidin S
Total Page：16
File Type：pdf, Size：1020Kb
The EMBO Journal Vol.16 No.14 pp.4174–4183, 1997 Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S Elena Conti1,2, Torsten Stachelhaus3, the peptide product. Each amino acid is activated by Mohamed A.Marahiel3 and Peter Brick1,4 adenylation of its carboxylate group with ATP and then transferred to the thiol group of an enzyme-bound phospho- 1Biophysics Section, Blackett Laboratory, Imperial College, pantetheine cofactor for possible modification and the 3 London SW7 2BZ, UK and Biochemie/Fachbereich Chemie, elongation reaction (Stachelhaus and Marahiel, 1995a; Philipps-Universita¨t Marburg, D-35032 Marburg, Germany Kleinkauf and von Do¨hren, 1996). 2 Present address: Laboratory of Molecular Biophysics, The cloning and sequencing of several peptide synthe- Rockefeller University, New York, NY10021, USA tase genes have revealed a conserved and ordered modular 4Corresponding 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 a In 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 phos- to 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 59-end of the grs phobic pocket with the carboxylate group interacting operon, encodes a 29 kDa protein homologous to fatty- with Lys517 and the a-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; Kra¨tzschmar et al., 1989). and indicates a conserved mechanism of nucleotide GrsA activates L-phenylalanine to the corresponding acyl- binding and substrate activation. adenylate and catalyses the inversion of configuration of Keywords: non-ribosomal peptide biosynthesis/peptide the amino acid. D-phenylalanine is then transferred to the synthetases/X-ray crystallography grsB gene product, gramicidin S synthetase 2 (GrsB), a 510 kDa polypeptide chain which sequentially activates proline, valine, ornithine and leucine and forms the peptide bonds in the elongation reaction, releasing the decapeptide Introduction (D-Phe-Pro-Val-Orn-Leu)2 after cyclization. A number of oligopeptides, some of which have important Each of the five modules in which the grs operon is medical and biotechnological applications, are produced organized encodes for highly conserved functional sub- by fungi and bacteria via a non-ribosomal mechanism. units. The major one is a 60 kDa fragment which recog- Peptides such as the cyclic gramicidin S and cyclosporin nizes a specific amino acid and catalyses the adenylation A, the lactone actinomycin, the branched bacitracin and of the amino acid carboxylate group with the α-phosphate the linear precursor of both penicillin and cephalosporin, of ATP. This adenylation subunit is conserved not only are synthesized by large multifunctional enzymes which within all known peptide synthetases, but also shares act as protein templates for the growing polypeptide chain. extensive sequence similarity with firefly luciferases and Peptide synthetases catalyse the repetitive activation and acyl CoA ligases. Common to all these enzymes is the condensation of the constituent amino acids to yield ATP-dependent activation of substrates as acyl adenylates. 4174 © Oxford University Press X-ray structure of gramicidin synthetase 1 On the other hand, the adenylation subunit shares no sequence homology with enzymes involved in the ribo- somal synthesis of polypeptides, despite the fact that the formation of aminoacyl-adenylates is chemically ana- logous in the two systems. Indeed, the crystal structure of firefly luciferase has indicated a structural framework unrelated to those of both class I and class II aminoacyl- tRNA synthetases (Conti et al., 1996). We report here the crystal structure of the phenylalanine- activating subunit of gramicidin synthetase 1 (PheA) in a ternary complex with phenylalanine and AMP. The struc- ture reveals the role of residues which are highly conserved in the superfamily of adenylate-forming enzymes. In addition, the presence of the substrate provides details of the amino acid specificity and allows a sequence-based comparison to be made with other peptide synthetases. A comparison of the structure with that of unliganded firefly luciferase reveals that both a domain rotation and a conformational change of a loop in the N-terminal domain must occur for luciferase to form an active complex with luciferin and ATP. Fig. 1. Ribbon diagram of the PheA molecule with the large N-terminal domain shown in blue and the small C-terminal domain in green. The disordered loop (residues 192–196) near the active site is coloured violet. The AMP (red) and phenylalanine (orange) ligands are Results and discussion drawn using a space-filling representation. The side chain of Lys517 on the loop that projects down from the C-terminal domain is drawn Crystal structure determination in green using a ball-and-stick representation. The crystal structure of PheA was determined by the multiple isomorphous replacement method, together with B6 in sheet B while strands A5–A6 correspond to strands real-space non-crystallographic symmetry averaging and B1–B2. refined against 1.9 Å resolution diffraction data to a The C-terminal domain (residues 429–530) includes crystallographic R-factor and R-free (Bru¨nger, 1992) of two helices which pack against one side of a three- 21.4% and 24.6% respectively. The model for 512 residues stranded antiparallel β-sheet E as well as an additional has good stereochemistry and includes phenylalanine and small sheet containing two β-strands. The polypeptide AMP bound at the active site. No interpretable electron chain at the C-terminus of the protein loops back towards density is present for the 16 N-terminal residues, the 33 the N-terminal domain and then packs against the C-terminal residues, nor for a loop containing residues remaining face of β sheet E (Figure 1). Residues at both 192–196. The two copies of the molecule in the asymmetric the N- and C-termini of the polypeptide chain project out unit have a very similar conformation: after superposition from the surface of the molecule and are relatively less the r.m.s. difference in the position of the main chain well ordered. atoms of residues 21–530 is 0.26 Å. Ligand binding Description of the overall structure The crystal structure shows unambiguous electron density The polypeptide chain folds into two compact domains for the ligands bound at the active site (Figure 4). In spite (Figure 1). There are very few direct protein–protein of the presence of Mg-ATP and phenylalanine in the interdomain contacts and instead the interactions between crystallization conditions, the electron density is not con- the structural domains are mediated by a network of sistent with the product of the activation reaction: the hydrogen bonds between the side chains of the protein phenylalanyl adenylate has been hydrolysed to the corres- and a sandwiched layer of ordered water molecules. The ponding amino acid and AMP. Substrate recognition is much larger N-terminal domain comprising residues 17– accomplished by an extensive network of hydrogen bonds 428 contains three subdomains: a distorted β-barrel and with a number of charged or polar amino acid residues. two β-sheets which pack together to form a five-layered Most of the protein residues involved in substrate recogni- αβαβα tertiary structure (Figure 2). Subdomain A contains tion are contributed by the large N-terminal domain. a six-stranded β-sheet and three helices formed by a single However, it is a charged residue of the C-terminal domain, segment of the polypeptide chain (residues 91–203) while the strictly invariant Lys517, which is involved in two a seventh strand is formed by an insertion in the β-barrel key polar interactions with both the amino acid and the subdomain (Figure 3).