Figure 1. Tyrocidine synthetase 1 (TycA) is the first component of the antibiotic tyrocidine biosynthetic system from bacterium Bacillus brevis. Full assembly consists of three NRPS, each composed of one (TycA), three (TycB), and six (TycC) modules. EXPLORING THE ROLE OF A CONSERVED MOTIF IN THE ADENYLATION DOMAIN OF A NON-RIBOSOMAL PEPTIDE SYNTHETASE FROM BACILLUS BREVIS V. Bučević-Popović, M. Šprung, B. Soldo, S. Orhanović, M. Pavela-Vrančić Faculty of Science, Department of Chemistry, Teslina 12, Split, CROATIA Adenylation Activity of A9 Core Motif Mutants. The aminoacyl- adenylate formation was first assayed by the amino-acid dependent ATP-[ 32 P]PPi exchange assay in the presence of natural substrate L -Phe (Fig. 5A). The rates of the exchange indicate that, with the exception of P490 mutants, single amino acid substitutions in A9 region have no significant influence on TycA adenylation activity. The effect of P490 mutation is especially evident in P490G enzyme, in which substitution of rigid proline with flexible glycine results in drastic reduction of enzymatic acitivtiy. Essentially the same results were obtained with complementary continuous spectophotometric pyrophosphate assay (Fig. 5B). In the absence of an activated amino acid acceptor (e. g. a holo-PCP domain), PPi release rate is limited by the slow leakage of amino-acyl adenylate from the active site (5). Although faster PPi release was measured in presence of non-cognate D -Phe (approximately fivefold), relative activities of A9 mutants compared to the wild type were comparable irrespective of the stereoisomer used. Introduction Nonribosomal peptide synthetases (NRPS) are highly sophisticated natural nanomachines that catalyze the synthesis of small peptides with antibiotic, immunosuppressant, and anticancer activities. NRPS usually contain one module for each amino acid incorporated into the final peptide product. Each module consists of several catalytic domains that catalyze the activation of specific amino acids (adenylation (A) domain), covalent thioester binding (peptidyl-carrier-protein (PCP) domain), formation of peptide bond (condensation (C) domain), and optionally, various substrate modifications (Fig. 1) (1). Adenylation (A) domain of NRPS catalyzes the two-step reaction of ATP-driven activation of amino acid and its transfer to the PCP domain. Together with the with acyl- and aryl-CoA synthetases and firefly luciferase, A- domains constitute the superfamily of adenylate-forming enzymes. It has recently been proposed that adenylate-forming enzymes use a 140° domain rotation to present opposing faces of the C-terminal subdomain to the active site for the different partial reactions (2) (Fig. 2). Sequence alignment of A-domains allowed identification of 10 ‘core motifs’ (A1-A10), most of which were assigned particular functions in substrate binding and/or catalysis, based on structural and mutagenesis studies. This study was aimed at elucidating the role of A9 core motif (Fig. 3), whose function has not been established so far, using tyrocidine synthetase 1 (TycA) from B. brevis as a model. Figure 2. Two-step reaction catalyzed by TycA adenylation domain. In the first half-reaction, L-Phe is converted into enzyme- bound L-Phe-adenylate (L-Phe- AMP). In the second step, the activated L-Phe is transferred to the thiol group of PCP-linked 4’-phosphopantethein cofactor. Homology models for TycA A- domain were derived by MODELLER using A-domain from gramicidin S synthetase 1 (PDB ID: 1amuA) for adenylate- and and D-alanyl carrier protein ligase for thioester-forming conformation (PDB ID: 3e7w) as templates. N-terminal subdomain is shown in yellow, C-terminal subdomain in blue. Several highly conserved motifs that form the active site are shown in red, while the motif comprising conserved Asp that serves as a hinge for domain rotation is shown in green. Due to subdomain movement, A9 motif (shown in dotted van der Waals surface) occupies different positions relative to the active site. Acknowledgements This work is supported by the grant from the Croatian Ministry of Science, Education and Sports References 1.S. A. Sieber, M. A. Marahiel, Chem Rev 105 (2005) A. M. Gulick, ACS Chem Biol 4 (2009) E. Pfeifer, M. Pavela-Vrančić, H. von Döhren, H. Kleinkauf, Biochemistry 34 (1994) R. Dieckmann, M. Pavela-Vrančić, H. von Döhren, H. Kleinkauf, J Mol Biol 288 (1999) V. Bučević-Popović, M. Pavela-Vrančić, R. Dieckmann, H. Von Döhren, Biochimie 88 (2006) U. Linne, A. Schafer, M. T. Stubbs, M. A. Marahiel, FEBS Lett 581 (2007) A. S. Reger, R. Wu, D. Dunaway-Mariano, A. M. Gulick, Biochemistry 47 (2008) 8016 Phe-AMP A Phe PCP E TycATycB C A Phe A Phe A Asn A Gln A Pro PCP CCCC E CCCC A Orn A Val A Tyr A Leu Te TycC module 1module 2 module 4 module 6 module 8 module 10 module 3 module 5 module 7 module 9 Figure 3. A9 core motif. Sequence alignment of representative A- domains showing the region surrounding A9 core motif. The sequences correspond to the following proteins: P09095, tyrocidine synthetase 1 from Bacillus brevis; Q01757, ACV synthetase from Streptomyces clavuligerus; P19828, AngR protein from Vibrio anguillarum; P11454, enterobactin synthetase component F from Escherichia coli; P0C062, gramicidin S synthetase 1 from Bacillus brevis; Q08787, surfactin synthetase C from Bacillus subtilis. A9 Results and Discussion Generation and Purification of A9 Mutants of TycA. To examine the role of A9 conserved motif in A-domain of TycA, a set of eight mutant enzymes carrying single amino acid substitutions was created. Five residues in A9 motif (Fig. 3), L484, P485, Y487, M488 and P490, were mutated to Ala or Ala and Gly, while S491 following immediately after A9 region was mutated to Arg. The wild type and mutant enzymes were expressed in E. coli and purified to homogeneity according to Pfeifer et al. (3). Two mutants, P490A and P490G revealed a significantly decreased solubility, indicating that P490 residue might play a structural role. Limited Proteolysis of TycA. To evaluate the effect of A9 mutation on protein conformation, we employed partial proteolytic digestion with trypsin (Fig. 4.). In agreement with previous work (4), tryptic cleavage of wild type TycA, produces four main fragments resulting from cleavage in linker regions between domains or subdomains. However, tryptic digest of P490 mutants, lack the largest fragment containing C-terminal subdomain. Fast proteolytic degradation indicates a less compact fold of C-terminal subdomain as a result of mutation. Figure 4. Patrial tryptic digestion of TycA. Proteolytic digestions of wild type and P490ATycA were performed in 50 mM, pH 7.5, at protein:protease ratio 50:1 (w:w). in the absence of substrates. In the presence of substrates (L-Phe and ATP), the rate of proteolysis was decreased but the proteolytic pattern remained the same. t (min) wt TycA t (min) P490A TycA A E PCP ACAC E E ANAN E ANAN E A SH A9 C-terminal subdomain rotation adenylate-forming conformation thioester-forming conformation A PhePhe-AMP ATPPP i A B Figure 5. Effect of mutations in A9 motif on TycA adenylation activity. Adenylation activity was measured by ATP-[ 32 P]PPi exchange assay (A) using L -Phe as a substrate and continuous spectrophotometric pyrophosphate assay (B) with either L -Phe or D -Phe. Reaction mixtures contained saturating substrate concentration (1 mM). Activities are expressed relative to the value measured with the wild type TycA. [CoA] (mM) Initial velocity (min -1 ) ● WT ■ L484A ○ P485A ● P485G □ Y487A ● M488A ■ P490A ■ S491R -adenylation domain- condensation domain -peptidiyl carrier protein - epimerisation domain -thioesterase domain AC Te PCP E PPi Release Activity in the Presence of Amino-acyl Acceptor. In order to monitor the complete A-domain catalyzed reaction, we attempted to measure acceleration of PPi release as a result of interdomain transfer of aminoacyl to TycA holo-PCP domain added in trans. TycA PCP domain was coexpressed together with Srf protein, catalyzing its in vivo posttranslational priming with 4’-phosphopantetein cofactor. Although, HPLC analysis (not shown) indicates that the majority of PCP protein is in its holo form, the acceleration of PPi release upon addition of holo-PCP was not observed. PPi release was also monitored in the presence of CoA, that may serve as a mimic of a 4’-phophopanthetein cofactor bound to PCP (6). CoA stimulates PPi release activity of TycA in presence of D -Phe (Fig. 6), while it has no effect when assayed in presence of TycA natural substrate, L -Phe. In the presence of natural substrate, enzyme probably adopts the conformation that is selectively suited for transfer of amino acid to holo-PCP. Whether slightly reduced ability to transfer activated amino acid to CoA in presence of non-cognate amino acid reflects the influence of mutations in A9 motif on the second half-reaction of A-domain catalytic cycle, remains to be further examined. Figure 6. PPi release rate in the presence of CoA. The most prominent increase was observed for S491R enzyme, designed to resemble post-A9 region of 4- chlorobenzoate:CoA ligase, which uses CoA as an acceptor cosubstrate in the second half-reaction (7). The increase of PPi release rate by CoA was smaller than in wild type enzyme for all of the other mutant proteins. Due to the low adenylation activity, P490G mutant was not assayed with CoA. The PPi release rate was assayed with D -Phe as amino acid substrate.