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• W1970386034 abstract "Article15 July 1997free access Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S Elena Conti Elena Conti Biophysics Section, Blackett Laboratory, Imperial College, London, SW7 2BZ UK Search for more papers by this author Torsten Stachelhaus Torsten Stachelhaus Biochemie/Fachbereich Chemie, Philipps–Universität Marburg, D-35032 Marburg, Germany Search for more papers by this author Mohamed A. Marahiel Mohamed A. Marahiel Biochemie/Fachbereich Chemie, Philipps–Universität Marburg, D-35032 Marburg, Germany Search for more papers by this author Peter Brick Corresponding Author Peter Brick Biophysics Section, Blackett Laboratory, Imperial College, London, SW7 2BZ UK Search for more papers by this author Elena Conti Elena Conti Biophysics Section, Blackett Laboratory, Imperial College, London, SW7 2BZ UK Search for more papers by this author Torsten Stachelhaus Torsten Stachelhaus Biochemie/Fachbereich Chemie, Philipps–Universität Marburg, D-35032 Marburg, Germany Search for more papers by this author Mohamed A. Marahiel Mohamed A. Marahiel Biochemie/Fachbereich Chemie, Philipps–Universität Marburg, D-35032 Marburg, Germany Search for more papers by this author Peter Brick Corresponding Author Peter Brick Biophysics Section, Blackett Laboratory, Imperial College, London, SW7 2BZ UK Search for more papers by this author Author Information Elena Conti1,2, Torsten Stachelhaus3, Mohamed A. Marahiel3 and Peter Brick 1 1Biophysics Section, Blackett Laboratory, Imperial College, London, SW7 2BZ UK 2Laboratory of Molecular Biophysics, Rockefeller University, New York, NY, 10021 USA 3Biochemie/Fachbereich Chemie, Philipps–Universität Marburg, D-35032 Marburg, Germany The EMBO Journal (1997)16:4174-4183https://doi.org/10.1093/emboj/16.14.4174 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The non-ribosomal synthesis of the cyclic peptide antibiotic gramicidin S is accomplished by two large multifunctional enzymes, the peptide synthetases 1 and 2. The enzyme complex contains five conserved subunits of ∼60 kDa which carry out ATP-dependent activation of specific amino acids and share extensive regions of sequence similarity with adenylating enzymes such as firefly luciferases and acyl-CoA ligases. We have determined the crystal structure of the N-terminal adenylation subunit in a complex with AMP and L-phenylalanine to 1.9 Å resolution. The 556 amino acid residue fragment is folded into two domains with the active site situated at their interface. Each domain of the enzyme has a similar topology to the corresponding domain of unliganded firefly luciferase, but a remarkable relative domain rotation of 94° occurs. This conformation places the absolutely conserved Lys517 in a position to form electrostatic interactions with both ligands. The AMP is bound with the phosphate moiety interacting with Lys517 and the hydroxyl groups of the ribose forming hydrogen bonds with Asp413. The phenylalanine substrate binds in a hydrophobic pocket with the carboxylate group interacting with Lys517 and the α-amino group with Asp235. The structure reveals the role of the invariant residues within the superfamily of adenylate-forming enzymes and indicates a conserved mechanism of nucleotide binding and substrate activation. Introduction A number of oligopeptides, some of which have important medical and biotechnological applications, are produced by fungi and bacteria via a non-ribosomal mechanism. Peptides such as the cyclic gramicidin S and cyclosporin A, the lactone actinomycin, the branched bacitracin and the linear precursor of both penicillin and cephalosporin, are synthesized by large multifunctional enzymes which act as protein templates for the growing polypeptide chain. Peptide synthetases catalyse the repetitive activation and condensation of the constituent amino acids to yield the peptide product. Each amino acid is activated by adenylation of its carboxylate group with ATP and then transferred to the thiol group of an enzyme-bound phosphopantetheine cofactor for possible modification and the elongation reaction (Stachelhaus and Marahiel, 1995a; Kleinkauf and von Döhren, 1996). The cloning and sequencing of several peptide synthetase genes have revealed a conserved and ordered modular organization. Each module encodes a functional building unit containing ∼1000 amino acids, which specifically recognizes a single amino acid. Within such a protein template-directed peptide biosynthesis, the occurrence and specific order of the modules in the genomic DNA dictate the number and sequence of the amino acids to be incorporated into the resulting oligopeptide. The modular arrangement of peptide synthetases closely parallels the multienzyme complexes responsible for the biogenesis of fatty acids and of the polyketide family of natural products. Furthermore, peptide synthetases, fatty-acid synthetases and polyketide synthetases all use enzyme-bound phosphopantetheine cofactors as acyl carriers, in a thiotemplate mechanism first proposed by Lipmann more than 20 years ago (Lipmann, 1971) and revised recently (Stein et al., 1996). In particular, the synthesis of the cyclic antibiotic gramicidin S has been studied in detail. Gramicidin S is produced by the Gram-positive bacterium Bacillus brevis and consists of two identical pentapeptides joined head to tail. It is synthesized by the multienzyme complex gramicidin S synthetase, which is encoded by the 19 kb grs operon that includes the genes grsA, grsB and grsT. The grsT gene, which is located at the 5′-end of the grs operon, encodes a 29 kDa protein homologous to fatty-acid thioesterases. The grsA gene product, gramicidin S synthetase 1 (GrsA) is a protein composed of 1098 amino acids (Hori et al., 1989; Krätzschmar et al., 1989). GrsA activates L-phenylalanine to the corresponding acyl-adenylate and catalyses the inversion of configuration of the amino acid. D-phenylalanine is then transferred to the 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 (D–Phe-Pro-Val-Orn-Leu)2 after cyclization. Each of the five modules in which the grs operon is organized encodes for highly conserved functional subunits. The major one is a 60 kDa fragment which recognizes a specific amino acid and catalyses the adenylation of the amino acid carboxylate group with the α-phosphate of ATP. This adenylation subunit is conserved not only within all known peptide synthetases, but also shares extensive sequence similarity with firefly luciferases and acyl CoA ligases. Common to all these enzymes is the ATP-dependent activation of substrates as acyl adenylates. On the other hand, the adenylation subunit shares no sequence homology with enzymes involved in the ribosomal synthesis of polypeptides, despite the fact that the formation of aminoacyl-adenylates is chemically analogous 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 structure 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. Results and discussion Crystal structure determination The crystal structure of PheA was determined by the multiple isomorphous replacement method, together with real-space non-crystallographic symmetry averaging and refined against 1.9 Å resolution diffraction data to a crystallographic R-factor and R-free (Brünger, 1992) of 21.4% and 24.6% respectively. The model for 512 residues has good stereochemistry and includes phenylalanine and AMP bound at the active site. No interpretable electron density is present for the 16 N-terminal residues, the 33 C-terminal residues, nor for a loop containing residues 192–196. The two copies of the molecule in the asymmetric unit have a very similar conformation: after superposition the r.m.s. difference in the position of the main chain atoms of residues 21–530 is 0.26 Å. Description of the overall structure The polypeptide chain folds into two compact domains (Figure 1). There are very few direct protein–protein interdomain contacts and instead the interactions between the structural domains are mediated by a network of hydrogen bonds between the side chains of the protein and a sandwiched layer of ordered water molecules. The much larger N-terminal domain comprising residues 17–428 contains three subdomains: a distorted β-barrel and two β-sheets which pack together to form a five-layered αβαβα tertiary structure (Figure 2). Subdomain A contains a six-stranded β-sheet and three helices formed by a single segment of the polypeptide chain (residues 91–203) while a seventh strand is formed by an insertion in the β-barrel subdomain (Figure 3). The β-sheet B contains eight strands, of which the first two (B1–B2) are formed by residues occurring before β-sheet A in the polypeptide chain, while the remaining six strands (B3–B8) and four helices form a contiguous polypeptide segment located before the β-barrel subdomain in the sequence. Strands 1–6 in the two β-sheets share a similar topology, with strands A1–A4 in sheet A corresponding to strands B3–B6 in sheet B while strands A5–A6 correspond to strands B1–B2. Figure 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 drawn using a space-filling representation. The side chain of Lys517 on the loop that projects down from the C-terminal domain is drawn in green using a ball-and-stick representation. Download figure Download PowerPoint Figure 2.Stereo diagram of the large N-terminal domain of PheA showing the bound ligands coloured as in Figure 1. The β-sheet A is on the left-hand side, β-sheet B is on the right-hand side, and the β-barrel is at the top of the figure. The N-terminus of the protein is at the top of the figure. The disordered loop (residues 192–196) near the active site is coloured violet. Download figure Download PowerPoint Figure 3.Topological arrangement of the secondary structural elements in PheA. The circles represent α-helices and the arrows β-strands. The strands have been numbered sequentially for each of the five β-sheets. Sheets A, B and the β-barrel C are in the N-terminal domain while sheets D and E are in the C-terminal domain. Download figure Download PowerPoint The C-terminal domain (residues 429–530) includes two helices which pack against one side of a three-stranded antiparallel β-sheet E as well as an additional small sheet containing two β-strands. The polypeptide chain at the C-terminus of the protein loops back towards the N-terminal domain and then packs against the remaining face of β sheet E (Figure 1). Residues at both the N- and C-termini of the polypeptide chain project out from the surface of the molecule and are relatively less well ordered. Ligand binding The crystal structure shows unambiguous electron density for the ligands bound at the active site (Figure 4). In spite of the presence of Mg-ATP and phenylalanine in the crystallization conditions, the electron density is not consistent with the product of the activation reaction: the phenylalanyl adenylate has been hydrolysed to the corresponding amino acid and AMP. Substrate recognition is accomplished by an extensive network of hydrogen bonds with a number of charged or polar amino acid residues. Most of the protein residues involved in substrate recognition are contributed by the large N-terminal domain. However, it is a charged residue of the C-terminal domain, the strictly invariant Lys517, which is involved in two key polar interactions with both the amino acid and the adenosine, fixing their position in the active site and clamping the C-terminal domain in a productive orientation. The lysine residue is located at the bottom of the large loop that projects down into the active site from the C-terminal domain (Figure 1). The key role played by this residue has been demonstrated by site-directed mutagenesis studies where the replacement of the corresponding lysine to a glutamine in the valine-activating domain of surfactin synthetase 1 results in the reduction of the reverse rate of adenylate formation by 94% (Hamoen et al., 1995). Figure 4.The difference electron density calculated with the AMP and phenylalanine ligands omitted from the model. The map was calculated using all data between 20.0 Å and 1.9 Å and contoured at 5 σ. Download figure Download PowerPoint Adenylate binding The adenylate is bound in a cleft present on the surface of the large N-terminal domain, between residues from β-sheet B and the β-barrel subdomains. A stereo diagram of the adenylate binding site is shown in Figure 5, while Figure 6 shows the hydrogen bonding interactions. The adenine moiety lies in a slot sandwiched between the side chains of Tyr323, Tyr425 and Ile348 on one side and the main chain atoms of residues 302–304 on the other. The binding of the base is mediated not only by the large area of hydrophobic and van der Waals interactions on the sides of the slot, but also by the hydrogen bonds of the N6 amino group with the main chain carbonyl oxygen of Ala322 and the side chain oxygen of Asn321. Hydrogen bonding to the exocyclic nitrogen of the adenine is the major specificity determinant by which the enzyme discriminates against guanine. No other ring nitrogen is in direct contact with the protein; of the possible hydrogen bonding interactions with the acceptor groups at positions 1, 3 and 7 of the purine ring, only N1 contacts a well-ordered water molecule (B = 22 Å2) which is in turn at 3.0 Å from the side chain nitrogen of Asn321. This pattern of interactions accounts for the catalytic activity displayed by the peptide synthetase in the presence of ATP analogues such as 7-deaza-ATP (Pavela-Vrancic et al., 1994). Figure 5.Stereo diagram showing the interactions made by the phenylalanine substrate and AMP (both in green) with PheA. The position of a possible magnesium ion is shown in yellow. Potential hydrogen bonding interactions are indicated by dotted lines. Download figure Download PowerPoint Figure 6.Schematic representation of the hydrogen bonding between PheA and the phenylalanine and AMP ligands. Download figure Download PowerPoint The ribose moiety is held in the C3′-endo conformation. The two hydroxyls of the sugar are involved in hydrogen-bonding interactions with the carboxylate of Asp413, which is a strictly invariant residue within the superfamily of adenylate-forming enzymes. The 2′ hydroxyl also forms a rather long (3.2 Å) hydrogen bond with the side chain of Tyr425 while Tyr323, which packs edge-on against the adenine ring, also hydrogen bonds to Asp413. In site-directed mutagenesis studies on the highly homologous tyrocidine synthetase 1, Gocht and Marahiel (1994) find that the replacement of the invariant aspartate by an asparagine residue reduces the ATP-PPi activity to 78% of that of the wild-type enzyme, while the substitution of a serine residue at this position reduces the activity to just 12%. Gramicidin synthetase 1 shows a much higher activity when ATP is replaced by 2′-deoxy-ATP (a 40% reduction) than when 3′-deoxy-ATP (85% reduction) is used in the ATP-PPi exchange reaction (Pavela-Vrancic et al., 1994). Although believed to be poor acceptors (Moodie et al., 1996), both the ribose O-4′ and O-5′ are hydrogen-bonded to the invariant Lys517. The α-phosphate of AMP has slightly weaker electron density, indicating that the binding site is more disordered. Interaction is with Thr326, a highly conserved residue in the superfamily of adenylate-forming enzymes, Thr190, also well conserved although replaced by a serine in the luciferases, together with the invariant Glu327. Glu327 points towards O1 of the phosphate and is bridged presumably by a magnesium ion, at 2.54 Å from the carboxylate and 2.26 Å from the phosphate. Phenylalanine binding The amino acid binding site of the peptide synthetase is a pocket with an entrance on the concave surface of the large domain near the intersection of the three subdomains. A stereo representation of the phenylalanine binding site of PheA is shown in Figure 7. The side chain of Asp235 and the main chain carbonyl oxygens of Gly324 and Ile330 are well placed to form hydrogen bonds with the α-amino group of the phenylalanine substrate. The aspartic acid is conserved in all peptide synthetases apart from the L-α-aminoadipate activating domain of ACV synthetase. In the PheA structure, Ile330 has dihedral angles (φ = 74°, ψ = −64°) outside the allowed regions of a Ramachandran plot (Ramakrishnan and Ramachandran, 1965). As is commonly observed in protein structures, the energetically unfavourable main chain dihedral angle is associated with a region of the molecule having a functional role (Herzberg and Moult, 1991). The α-carboxylate group of the substrate amino acid is stabilized by an electrostatic interaction with the invariant Lys517 from the C-terminal domain. Figure 7.Stereo diagram showing the side chains of PheA that line the specificity pocket for the phenylalanine substrate (in green). The unlabelled Cys331 is hidden behind the substrate. The main chain carbonyl groups of residues Ile334 and Gly324 have been included. Potential hydrogen bonding interactions are indicated by dotted lines. Download figure Download PowerPoint The specificity pocket for the phenylalanine side chain is surrounded by residues from the strands and a helix associated with β-sheet B (Figures 2 and 7). The pocket is lined at the bottom by the indole ring of Trp239, on one side by Ala236, Ile330 and Cys331 and on the opposite side by Ala322, Ala301 and Thr278. The two sides of the pocket are appropriately separated to accommodate an aromatic residue but at one end of the pocket (towards the viewer in Figure 7) there is a water-filled channel that connects with the solvent. The phenylalanine binding pocket can accommodate both stereoisomers of the amino acid with no significant change in the protein conformation. In the 2.0 Å refined crystal structure of a ternary complex containing D-phenylalanine and AMP, the polar interactions between the ligand and the protein are identical to those in the L-phenylalanyl complex but the benzene ring of the side chain is rotated by 30° about an axis perpendicular to the plane of the ring. This rotation leads to a 1.3 Å displacement in the relative position of the Cβ atoms, but the Cα atoms are within 0.5 Å of one another and the oxygen atoms that interact with the side chain of Lys517 are within 0.26 Å. Over 50 sequences are now known for the amino acid activating modules of peptide synthetases and although the enzymes differ in their substrate specificity, they show extensive regions of sequence similarity to PheA. Of the modules listed in Table I, the percentage of identical residues ranges from 26% for module 3 of the HC-toxin synthetase to 56% for the phenylalanine-activating module of tyrocidine synthetase. With this level of sequence identity, the main chain conformation of the enzymes is likely to be very similar and the differing substrate specificities will be mainly determined by the nature of the amino acids lining the substrate binding pocket. Table I lists these amino acids for several different peptide synthetases. A number of enzymes contain charged residues near the binding pocket and for the activation of substrates with charged side chains such as ornithine, aspartate and glutamate, there are protein side chains at either position 239 or 278 with opposite charge. The first module of ACV synthetase adenylates the δ-carboxylate rather than the α-carboxylate of the L-α-aminoadipate side chain, and it is possible that the α-amino and α-carboxylate groups of the substrate bind at the bottom of the pocket and interact with the arginine at position 239 and the glutamate at position 322. This mode of binding could explain the absence of an aspartate residue at position 235 to interact with the α-amino group of the amino acid substrate. It can also be seen from Table I that charged residues are sometimes present near the pocket in enzymes that activate amino acids with neutral side chains, while synthetase modules that are specific for the same substrate can have different amino acids around the substrate binding pocket. Table 1. Identity of residues lining the substrate binding pocket in various peptide synthetases Synthetase (module number) Substrate (%) Identity with PheA Residue (PheA numbering) 236 239 278 299 301 322 330 331 Gramicidin 1: PheA Phe A W T I A A I C Tyrocidine 1 Phe 56 T F T I A A I C Surfactin 1 (2) Leu 37 A F M F G M V F Surfactin 1 (3) & 2 (3) Leu 36–37 A W F T G N V V Gramicidin 2 (4) Leu 42 G A Y T G E V V Cyclosporin (2,3,8,10) Leu 30–31 A W L Y G A V M Gramicidin 2 (2) Val 40 A F W I G G T F ACV (3) Val 28 F E S T A A V Y Cyclosporin (4,9) Val 30–31 A W M F A A I/V L Surfactin 2 (1) Val 38 A F W I G G T F ACV (2) Cys 33 H E S D V G I T HC-toxin (1) Pro 27 I A V I T V L I Gramicidin 2 (1) Pro 41 C K S I A H V V Cyclosporin (1) D-Ala 30 L W F L I A V V Cyclosporin (11) Ala 30 V F I Y A A I L HC-toxin (2) Ala 27 A G G C A M V A HC-toxin (3) Ala 26 L L F G I S V L Cyclosporin (7) Gly 31 I Q M F V A M Q ACV (1) Aad 32 P R N I V E F V Gramicidin 2 (3) Orn 42 V G E I G S L I Cyclosporin (5) Bmt 31 A W T Y G G V I Cyclosporin (6) Abu 30 A W F H A V A Y Surfactin 1 (1) Glu 34 A K D L G V V D Surfactin 2 (2) Asp 33 L T K V G H I A Unusual amino acids: Aad, L-2-aminoadipic acid; Abu, α-amino butyric acid; Bmt, (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine. References for sequences: Tyrocidine synthetase 1 (Weckermann et al., 1988), HC-toxin synthetase (Scott-Craig et al., 1992), surfactin synthetase 1 (Fuma et al., 1993), surfactin synthetase 2 (Cosmina et al., 1993), ACV synthetase from Penicillium chrysogenum (Smith et al., 1990), gramicidin synthetase 1 (Krätzschmar et al., 1989), gramicidin synthetase 2 (Hori et al., 1989), cyclosporin synthetase (Weber et al., 1994). Modules 2,3,4,5,7,8 and 10 of cyclosporin synthetase contain a 447 residue insertion between β-strand E2 and the following helix in the C-terminal domain which catalyses the N-methylation of the amino-acid. E.Conti et al Comparison with the structure of firefly luciferase As was anticipated from the 16% sequence identity and the presence of short highly conserved amino acid motifs equidistantly separated in the two sequences (Figure 8), the topology of each of the structural domains of PheA is very similar to that observed in the crystal structure of the unliganded firefly luciferase (Conti et al., 1996). PheA is slightly smaller, is missing an extra strand in sheet A, an α-helix near the C-terminus and, where the luciferase enzyme has a β-strand at the N-terminus of the molecule, PheA has an α-helix. The most striking difference between the two crystal structures is the relative orientation of the N- and C-terminal domains. When compared with the luciferase structure, the C-terminal domain of PheA is rotated by 94° relative to the N-terminal domain and is 5 Å closer to it (Figure 9). The loops 436–440 and 524–528 (luciferase numbering) near the domain interface which are disordered in luciferase have well-defined electron density in the PheA crystal structure. Optimal superposition of the N-terminal domains of the two enzymes results in an r.m.s. separation of 1.5 Å for the 287 pairs of equivalent Cα atoms with a separation <3 Å (Figures 8 and 9), while superposition of the C-terminal domains gives an r.m.s. separation of 1.42 Å for the 77 Cα atoms within 3 Å. The structure of PheA in a binary complex with Mg-AMP-PNP in which the PNP moiety is disordered (data not shown) reveals a similar domain orientation to the one observed for the ternary complex, indicating that the presence of the amino acid is not required to obtain this productive conformation of the protein. Figure 8.Structurally based amino acid sequence alignment of PheA (top row) with firefly luciferase (Conti et al., 1996) (bottom row). Lower-case characters indicate residues omitted from the molecular models. Asterisks indicate pairs of residues for which the separation of the Cα atoms is <3 Å after separate optimal superposition of either the N-terminal or the C-terminal domains of the molecular structures. The position of secondary structural elements in PheA determined using the algorithm of Kabsch and Sander (1983) are indicated above the sequence. The β-strands are labelled as in Figure 3. Residues that are highly conserved within the superfamily of adenylate-forming enzymes are shaded. Download figure Download PowerPoint Figure 9.Comparison of the crystal structures of (A) PheA and (B) firefly luciferase. The larger N-terminal domains in the lower portion of the figures are viewed in a similar orientation. For each domain, the shaded regions of the structure corresponding to residues for which the separation of the Cα atoms is <3 Å after optimal superposition. The relative orientation of the smaller C-terminal domains are related by a 94° rotation about an axis represented by a dashed line in each figure. Download figure Download PowerPoint There is a major difference in the entrance to the cavity which in PheA is occupied by the amino acid ligand. Although most of the main chain atoms of the two enzymes superpose well around the active site, the loop containing residues 314–319 in firefly luciferase (residues 300–305 in PheA) has a different conformation and obstructs both the entrance to the large water-filled luciferin binding pocket and the binding of the adenine ring of the nucleotide. A significant conformational change of this loop must occur in the firefly luciferase molecule to accommodate the binding of the ligands. The mutation of the highly conserved glycine residue in this loop (Gly302 in PheA) to a glutamic acid in the valine activating domain of GrsB results in the complete loss of PPi-ATP exchange activity (Saito et al., 1995). The addition of a side chain at this position is likely to compromise the movement of the loop and cause a change in the conformation of the main chain as the dihedral angles for Gly302 (φ = 99°, ψ = −41°) are unfavourable for non-glycine residues. Signature sequence The most highly conserved sequence of amino acids in the superfamily of adenylate-forming enzymes involves residues 190-TSGTTGNPKG-199 which in the PheA structure form a loop between β-strands 5 and 6 in subdomain A. The absence of significant electron density for residues 192–196 implies that the central residues of the loop have conformational flexibility. The corresponding loop is also disordered in the crystal structure of the firefly luciferase. These residues are not involved in the binding of the AMP moiety; their position with respect to the AMP binding site suggests that they are likely to interact with the pyrophosphate leaving group (Figure 5)Gycine-rich loops are often present in ATP- and GTP-binding proteins and are generally found to form an anion hole which accommodates the phosphate of the nucleotide (Pai et al., 1989; Knighton et al., 1991). As discussed above, Thr190 at the beginning of the conserved peptide interacts with the α-phosphate, while the side chain of Lys198 is poorly ordered and projects into solvent. The importance of Lys198 in the PPi-ATP exchange reaction has been demonstrated by site-directed mutagenesis of the corresponding lysine residue in tyrocidine synthetase 1 (Gocht and Marahiel, 1994). Both Lys198 and the invariant arginine at position 428 (Figure 5) are probably involved in coordinating the pyrophosphate group, while the lysine at position 517 is likely to stabilize the negatively charged pentavalent transition state in a manner analogous to that of the invariant arginine of class II aminoacyl-tRNA synthetases. The arginine residue forms an ion pair with the α-carboxylate moiety of the amino acid substrate but interacts with the α-phosphate when the aminoacyl-adenylate intermediate is formed (Onesti et al., 1995). The knowledge of the residues involved in forming the substrate specificity pocket provided by this study combined with the wealth of amino acid sequence information available for other adenylate-forming domains provides a structural basis for understanding the specificity of peptide synthetases. These findings should allow the manipulati" @default.
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• W1970386034 title "Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S" @default.
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