©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Modular Structure of Peptide Synthetases Revealed by Dissection of the Multifunctional Enzyme GrsA (*)

(Received for publication, September 16, 1994; and in revised form, December 28, 1994)

Torsten Stachelhaus Mohamed A. Marahiel (§)

From the From Biochemie/Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Straße, 35032 Marburg, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Analysis of the primary structure of peptide synthetases involved in non-ribosomal synthesis of peptide antibiotics revealed a highly conserved and ordered domain structure. These functional units, which are about 1000 amino acids in length, are believed to be essential for amino acid activation and thioester formation. To delineate the minimal extension of such a domain, we have amplified and cloned truncated fragments of the grsA gene, encoding the 1098-amino acid multifunctional gramicidin S synthetase 1, GrsA. The overexpressed His(6)-tagged GrsA derivatives were affinity-purified, and the catalytic properties of the deletion mutants were examined by biochemical studies including ATPdependent amino acid activation, carboxyl thioester formation, and the ability to racemize the covalently bound phenylalanine from L- to the D-isomer. These studies revealed a core fragment (PheAT-His) that comprises the first 656 amino acid residues of GrsA, which restored all activities of the native protein, except racemization of phenylalanine. A further deletion of about 100 amino acids at the C-terminal end of the GrsA core fragment (PheAT-His), including the putative thioester binding motif LGGHSL, produced a 556-amino acid fragment (PheA-His) that shows a phenylalanine-dependent aminoacyl adenylation, but almost no thioester formation. A 291-amino acid deletion at the C terminus of the native GrsA, that contains a putative racemization site resulted in complete loss of racemization ability (PheATS-His). However, it retained the functions of specific amino acid activation and thioester formation. The results presented defined biochemically the minimum size of a peptide synthetase domain and revealed the locations of the functional modules involved in substrate recognition and ATP-dependent activation as well as in thioester formation and racemization.


INTRODUCTION

Non-ribosomally synthesized peptides produced by several bacterial and fungal species belong to a diverse family of natural products that includes antibiotics, immunosuppressants, plant and animal toxins, and enzyme inhibitors. The manifold biological activities are emphasized by numerous structural variations of these peptides. The structures are linear, cyclic, or branched, and they contain D-, hydroxy, and N-methylated amino acids as well as other amino acid (aa) (^1)constituents that can undergo extensive modifications, including acylation and glycosylation(1, 2, 3) . The synthesis of these peptides is accomplished by large multienzyme complexes that possess a multidomain structure and employ the thiotemplate mechanism(4) . The reaction sequence involves ATP-dependent amino acid activation, followed by transferring the acyl adenylate to specific thiol groups and formation of a carboxyl thioester-bound substrate. The elongation reaction is assisted by an enzyme-bound cofactor, the 4`-phosphopantetheine(1, 2, 3, 4) . Recent biochemical and genetic studies suggested a model for the elongation reaction that assumes the involvement of multiple cofactors of the 4`-phosphopantetheine type. One cofactor for each amino acid activating domain was suggested, rather than the presence of a centrally located sole cofactor(5, 6) . These cofactors may facilitate the ordered shift of the carboxyl thioester-activated amino acids between the domains that assemble the multienzyme, resulting in the formation of a peptide chain with a defined sequence(7, 8, 9) . Termination of this enzyme-catalyzed peptide synthesis is induced by the release of the thioester-bound peptide by cyclization, hydrolysis, or specific transfer to a functional group.

Sequence comparison of the deduced primary structure of an increasing number of peptide synthetases revealed the multidomain arrangement of these enzymes(7) , whose specific linkage order has been shown to define the sequence of the incorporated amino acid residues(4, 7, 9, 10, 11, 12, 13) . Protein chemical studies have identified proteolytic fragments with molecular masses of about 110-120 kDa able to activate an individual amino acid(10, 14, 15, 16, 17) . These fragments seemed to correspond with the characterized activating spots, defined by Lipmann (18) .

On the other hand, sequence comparisons revealed for a diverse number of multifunctional peptide synthetases the presence of homologous domains of about 600 amino acids in length(2, 7) . These regions contain six highly conserved motifs, designated core sequences (see Fig. 1b), which are typical for a superfamily of adenylate- and thioester-forming enzymes(7) . Previous studies, including site-directed mutagenesis and photoaffinity labeling with ATP analogous, have shown the involvement of the core sequences 2-5 in ATP binding and hydrolysis(19, 20, 21, 22) . They also suggested the association of core 6 in covalent binding of the substrate amino acid(5, 20, 23) . These results hint that only a conserved domain, rather than a complete peptide synthetase, performed the specific activation of a cognate amino acid. However, although core motifs were suggested to be essential for this activity, it was never shown biochemically that only the conserved domain could activate an individual amino acid.


Figure 1: Organization of the gramicidin S biosynthesis operon and structural architecture of GrsA and derivatives. a, organization of the entire grs operon, comprising the genes grsT, grsA, and grsB, and the location of the gsp gene, associated with the gramicidin S biosynthesis system(40) , are shown. The linkage order of the domains (black boxes, adenylation module; shaded area, thioester formation module) has been proposed to define the sequence of the incorporated amino acid residues in the peptide product(2) . b, several highly conserved motifs of peptide synthetases were identified within gramicidin S synthetase 1, GrsA. The localization of putative adenylate-forming, thioester-forming, and racemase units are shown. c, the structural architecture and the restored core motifs of the deletion mutants PheATS-His, PheAT-His, and PheA-His are shown. The GrsA derivative PheATS-His is devoid of the putative racemase region, and the constructs PheAT-His and PheA-His possess the extension of the conserved regions of peptide synthetase and adenylate-forming enzymes, respectively(7) . d, table summarizes the highly conserved core motifs, their consensus sequences, and their putative functions within ATP-dependent amino acid activation.



Based on sequence alignments and a limited number of biochemical studies, two types of domains have been characterized within bacterial and fungal peptide synthetases. Type I comprises about 1100 amino acids and bears the functions of amino acid activation and thioester formation(1, 2, 3) , whereas type II carries in addition an insertion of about 430 amino acids, which may function as a N-methyltransferase module(12, 13) . The latter type of domain was only found in fungal peptide synthetases involved in the synthesis of the cyclic peptides enniatin and cyclosporin. Both synthetases catalyze the incorporation of N-methylated amino acids. Furthermore, the sequence analysis revealed a conserved region located to the C-terminal end of type I domains that activate D-configurated amino acids. Therefore, this region was designated as a putative racemization module(8) .

In order to understand the structure/function relationship of peptide synthetases and to determine the arrangement of putative modules facilitating substrate adenylation, thioester formation and racemization, we used GrsA as a model enzyme. The grsA gene encodes the 1098-amino acid residue gramicidin S synthetase 1 (GrsA), which catalyzes the first step in the biosynthesis of the cyclic decapeptide gramicidin S(4, 24) . It activates L-phenylalanine and racemizes it to the D-isomer. Therefore, GrsA represents a model for a type I domain that contains all integrated functions within a single polypeptide chain with a molecular mass of 126.7 kDa. We define in this work, by deletion studies, the minimal size of an amino acid activating domain and determine the locations of integrated modules involved in ATP-dependent acyladenylation, thioester formation, and epimerization.


EXPERIMENTAL PROCEDURES

PCR Amplification and DNA Manipulations

We used the pQE60 His(6) tag fusion vector, purchased from Qiagen (Hilden, Germany), for cloning of the truncated grsA fragments. pQE60 is a derivative of pDS described by Bujard et al.(25) and contains information required for replication as described for pACYC184 (26) , the T5 promotor, a ribosome binding site, a His(6) tag, a small multiple cloning site, and a transcriptional terminator(27) . The vector allowed the expression of the genes from the authentic ATG codon, but required the modification of the sequences around that codon to form a NcoI restriction site. It was also necessary to create a BamHI site at the 3` end, because the inserts had to be ligated in-frame with the His(6) tag included in the vector. To fit these requirements, the insert sequences could be modified using 5`-modified PCR primers to generate the terminal restriction sites needed. The sequences of the oligonucleotides, which were synthesized by Dr. Michael Krause (Mikrochemische Einheit, FB20, Philipps-Universität Marburg, Germany), were as follows (underlined, modified sequences; bold, restriction sites): oligo 5`-Phe-NcoI: 5`-ATA TCC ATG GTA AAC AGT TCT AAA AG-3`; oligo 3`-PheA-BamHI: 5`-TCT CGG ATC CTA ATA CAT CCT GCC AG-3`; oligo 3`-PheAT-BamHI: 5`-ATC GGA TCC ATT TGG TCT ATA CAA C-3`; and oligo 3`-PheATS-BamHI: 5`-ATA GGA TCC TAA TTC AAT AGA CCA GTC C-3`.

The amplification of the grsA fragments was performed using Deep Vent(R)® DNA polymerase, 10 times reaction buffer from New England Biolabs (Schwalbach, Germany), and different pairs of primers. The reaction conditions were: 0.2-20 ng of chromosomal DNA from Bacillus brevis ATCC 9999, 1-10 pmol of primers (equimolar), 300 µM deoxyribonucleoside triphosphates (dNTPs), 10 mM potassium chloride, 10 mM ammonium sulfate, 20 mM Tris/HCl, pH 8.8 at 25 °C), 6 mM magnesium sulfate, 0.1% Triton X-100, and 1 unit of Deep Vent(R)® DNA polymerase in a total volume of 100 µl(28) .

The PCR products were purified using the QIAquick-spin PCR purification kit as described by the manufacturer's protocol (Qiagen, Hilden, Germany). Standard procedures were used for the digestion with restriction enzymes, the cloning of the DNA fragments, and the preparation of the transformed plasmid DNA(28) . We also inspected both the ATG and the His(6) tag fusion sites between vector and truncated grsA fragments by sequencing using the chain termination method of Sanger et al.(29) . The sequences of the primers used, which were also synthesized by Dr. M. Krause, were as follows: oligo 5`-promotor: 5`-GGC GTA TCA CGA GGC CC-3`; 3`-His tag: 5`-ACG CCC GGC GGC AAC CG-3`.

Expression of the His(6)-tagged GrsA Derivatives

Modified expression plasmids containing the amplified grsA fragments were transformed in Escherichia coli M15(pREP4)(30) . The transformants were used to inoculate 2 times YT medium (31) supplemented with ampicillin (100 µg/ml), kanamycin (25 µg/ml), and magnesium chloride (10 mM). Cells were grown at 30 °C with moderate shaking until A reached 1.5-1.8. IPTG was added to a final concentration of 1.5 mM, and cultures were grown for an additional 3 h. The extent of expression was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) using the method of Laemmli(32) .

Enzyme Purification

All operations were carried out at 4 °C except the chromatography, which was performed at room temperature. Cells were collected by centrifugation for 15 min at 6,000 times g, and resuspended in sonication buffer (50 mM sodium phosphate, pH 8.0 at 23 °C, 300 mM sodium chloride) at 3 volumes/g of wet weight. Subsequently, the samples were lysed by freezing, thawing, and sonication using a Branson Sonifier at middle output. The cell debris was pelleted by centrifugation at 40,000 times g for 30 min, and the supernatant obtained was directly applied to a Ni-charged chelating Superose HR 10/2 column, previously equilibrated with sonication buffer. Both the immobilized metal ion affinity chromatography column and the fast performance liquid chromatography system were purchased from Pharmacia LKB Biotechnology Inc. (Freiburg, Germany). The proteins were eluted by applying imidazole gradients ranging over 0-250 mM in sonication buffer. Samples were analyzed by SDS-PAGE (10%), pooled, dialyzed against assay buffer (50 mM sodium phosphate pH 7.8 at 4 °C, 10 mM magnesium chloride, 2 mM dithioerythritol, and 1 mM EDTA), and measured using the procedure of Bradford (33) .

ATP-PP(i) Exchange

Essentially, ATP-PP(i) exchange of the GrsA derivatives was measured as described by Lee and Lipmann(34) . The label, tetrasodium [P]pyrophophate (16.06 Ci/mmol), was purchased from DuPont NEN (Bad Homburg, Germany). An assay mix contained 20 pmol of enzyme, 0.02-0.2 mM ATP, 0.02-0.2 mMD-Phe, 50 mM sodium phosphate, pH 7.8 at 23 °C, 1 mM dithioerythritol, 0.1 mM EDTA, 1 mM magnesium acetate, 0.1 mM tetrasodium pyrophosphate, and 0.15 µCi of [P]pyrophosphate in a total volume of 100 µl. Determination of the substrate specificity was performed under saturating conditions with 0.2 mM of the cognate and non-cognate amino acids, respectively.

Thioester Formation and Cleavage

Covalent binding of the substrate amino acid phenylalanine was tested as described previously by Gocht et al.(20) . L-[U-^14C]Phe (474 mCi/mmol) was purchased from Amersham/Buchler (Braunschweig, Germany). Each assay mix contained 0.1 nmol of enzyme, 0 or 2 mM ATP, 50 mM sodium phosphate, pH 7.8 at 23 °C, 1 mM dithioerythritol, 0.1 mM EDTA, 10 mM magnesium chloride, and 2 µCi of [^14C]Phe in a total volume of 100 µl. The reactions were allowed to proceed for 30 min at 37 °C. They were stopped by addition of 2 ml of ice-cold 7% trichloroacetic acid (TCA), and further incubated on ice for 30 min. Trichloroacetic acid-precipitated proteins were collected on glass fiber filters (GF-92; Schleicher & Schuell, Dassel, Germany), and washed with an excess of trichloroacetic acid. After addition of 5 ml of scintillation mixture (Rotiszint Eco Plus; Roth, Karlsruhe, Germany) retarded enzyme-bound [^14C]Phe was counted in a 1900CA Tri-Carb liquid scintillation analyzer (Packard).

Cleavage of the Thioesterified Phenylalanine

The cleavage of the enzyme-phenylalanine complexes was done as described by Ullrich et al.(35) . Trichloroacetic acid-precipitated complexes were washed five times with trichloroacetic acid and two times with ethanol to remove any traces of unbound label. The vacuum-dried pellets were suspended in 100 µl of cleavage solution (80% formic acid and 6% hydrogen peroxide) and incubated at 56 °C for 30 min. The reaction mixtures were dried again, extracted with 50% ethanol several times, and subjected to thin-layer chromatography (TLC) for analysis.

Thin-layer Chromatography

Analysis of protein-[^14C]Phe cleavage products was done by chiral plate high performance thin-layer chromatography (2.5 cm times 10-cm chiral high performance thin layer chromatography plates; Merck, Darmstadt, Germany) with solvent system A (methanol-water-acetonitrile; 1:1:4)(23) . The label was identified by autoradiography and by scanning on a Berthold LB2723 thin-layer scanner II.


RESULTS

Amplification and Cloning of the grsA Fragments

To delineate the minimal extension of an amino acid activating domain, we have amplified truncated fragments of the grsA gene. As shown in Fig. 1, we chose the 3` end oligonucleotides for the PCR in consideration of structural conservations found in peptide synthetases. The first fragment amplified comprises 2421 base pairs and encoded the 92.5-kDa mutant PheATS-His (Fig. 1c). It carries a deletion of 291 aa at the C-terminal end of the native peptide synthetase GrsA (1098 aa). This protein is devoid of a putative racemization module. The second amplified grsA fragment of 1968 base pairs encoded the 76.2-kDa deletion mutant PheAT-His. This construct, comprising 656 aa, reduced the primary structure of the intact GrsA protein to a conserved region, typical for adenylate- and thioester-forming enzymes. Also constructed was a third GrsA deletion. Here an additional 100 aa from the C-terminal end of PheAT-His was removed, resulting in the 556-aa (64.3-kDa) fragment PheA-His. This GrsA mutant is devoid of a putative thioester binding site and possesses the primary structure of a superfamily of adenylate-forming enzymes(7) . Fig. 1c summarizes the extent of the GrsA deletion mutants derived.

Amplification of the grsA fragments encoding the multienzyme derivatives, was performed as described under ``Experimental Procedures.'' The DNA fragments obtained were purified and cloned as described. In addition, each mutant plasmid was confirmed by sequencing the junctions between vector and inserted grsA fragments.

Expression and Purification of Truncated GrsA Derivatives

High level expression of the GrsA derivatives was obtained, using freshly transformed E. coli cells containing pREP4 and pQE60 derivatives. Optimization of the fermentation parameters of E. coli strain M15 with respect to the culture medium, the production time, and the concentration of IPTG revealed the conditions described under ``Experimental Procedures.'' A very important factor in the solubility of the expressed recombinant GrsA derivatives was the addition of magnesium chloride. We found that a concentration of 10 mM Mg in the culture medium increased the yield of soluble protein from 10% to more than 75%, constituting magnesium as a cofactor of peptide synthetases during ATP hydrolysis. Using optimized conditions, we found high level expression with a predominant soluble cytoplasmic location of the GrsA derivatives (Fig. 2a).


Figure 2: Expression and purification of the GrsA derivatives. Samples of the expression and purification of the deletion mutants were analyzed on 10% SDS-polyacrylamide slab gels (see ``Experimental Procedures''). a, Coomassie-stained gel showing the total cell extracts of E. coli M15(pREP4/pPheATS-His) at t(0) (lane 1) and 3 h after induction with IPTG (lane 2). Lane 3 shows the dialyzed PheATS-His pool after affinity purification on chelating Superose. b, the protein pools of PheATS-His (lane 1), PheAT-His (lane2), and PheA-His (lane3) after immobilized metal ion affinity chromatography are shown.



The purification of the expressed proteins was performed as described under ``Experimental Procedures.'' Using these optimized conditions, we obtained the recombinant proteins to near homogeneity. Fig. 2b shows the SDS-PAGE of the purified protein pools. The gel revealed molecular masses of 91, 70, and 62 kDa for the truncated GrsA deletion mutants PheATS-His, PheAT-His, and PheA-His, respectively. These data correspond to the values deduced from the DNA sequences. The protein pools were dialyzed against assay buffer, and protein concentration was determined.

ATP-PP(i) Exchange

The first step of amino acid activation during non-ribosomal peptide biosynthesis is the ATP-dependent substrate activation as acyladenylate(36) . We determined the influence of the introduced C-terminal deletions in GrsA on Phe-dependent ATP-PP(i) exchange. As shown in Table 1, only slight alteration in exchange activities were observed for the GrsA deletions, when compared with the native GrsA protein. The deletion of a putative racemase module in mutant PheATS-His induced the activity to nearly 120% of the wild type level for L-Phe-dependent ATP-PP(i) exchange. The deletions in GrsA derivatives PheAT-His and PheA-His caused no reduction in exchange activities and revealed normal levels for the L-Phe-dependent reaction. Interestingly, even the 556-aa construct PheA-His, possessing nearly half the size of GrsA (1098 aa), restored the same adenylation activity as the wild type protein (Table 1).



The substrate specificity of GrsA deletions was investigated with respect of the cognate amino acid phenylalanine, as well as of the non-cognate amino acids that comprise the peptide antibiotic gramicidin S and the Phe analogue 3-phenylpropionic acid. These studies were performed using saturating concentrations for all amino acids tested. As shown in Fig. 3, the amino acid-dependent activation revealed for all deletion mutants high exchange activities for D- and L-phenylalanine and almost no activation in the case of the Phe-carboxyl acid analogue 3-phenylpropionic acid, proline, valine, ornithine, and leucine. The highest nonspecific exchange activities obtained for the non-cognate hydrophobic amino acids L-Val and L-Leu were achieved for the mutant PheAT-His. This construct also revealed a slight reduction in D-Phe-dependent exchange activity. The slightly reduced specificity of the GrsA mutant PheAT-His was significant, although the reason for remains unclear, because the smaller fragment, PheA-His, restored high specificity for the cognate amino acids D- and L-phenylalanine. Nevertheless, this is the first clear indication that the core activities involving amino acid recognition and the ATP-dependent activation are located within the first 556 aa residues of the multifunctional peptide synthetase GrsA.


Figure 3: Amino acid specificity of the ATP-PP(i) exchange reaction of the GrsA derivatives. The ATP-PP(i) exchange reaction was measured for the three deletion mutants in dependence on the cognate amino acid phenylalanine, the Phe analogue 3-phenylpropionic acid (3-PPA), and the amino acid residues that comprise the peptide antibiotic gramicidin S (Pro, Val, Orn, and Leu, respectively). The highest exchange activity of each mutant was defined as 100%, and the values obtained for the other amino acid were settled with those.



Binding Affinities of the Truncated GrsA Derivatives

We investigated the binding affinities of the mutant GrsA proteins and the substrates ATP and phenylalanine, respectively. The kinetic constants (K(m)) were determined from Lineweaver-Burk plots in dependence on the concentration of both reaction partners (37) (data not shown). Plots obtained at different substrate concentrations intersect at abscissa values of -1/K(m). The kinetic constants determined for wild type GrsA and mutants are summarized in Table 1.

All deletion mutants bind the substrates ATP and phenylalanine with nearly the same affinity as the wild type protein. The K(m) values obtained also correspond to those determined previously for the four GrsB domains and TycA(20, 38) . Limited deviations in the binding affinities for ATP were observed. For the deletion mutants PheATS-His to PheA-His, the range was between 1.1 and 0.8 mM, indicating a slight increase in the ATP affinity for the shorter fragments. For the cognate amino acid phenylalanine, a 20-fold higher binding affinity could be detected for the GrsA deletions, and almost identical K(m) values were determined (60 µM; Table 1). Furthermore, the points of intersection within the different plots are located in the upper left quadrant, indicating a random binding of the substrates on GrsA and derivatives as shown previously(38) . The substrates ATP and Phe associates independently with the protein, and even if both ATP and phenylalanine are linked, the formation of the acyladenylate can be performed.

Covalent Binding of Phenylalanine

The second step of amino acid activation during non-ribosomal peptide synthesis accomplishes covalent binding of the carboxyl-activated amino acid as thioester (36) . As reported, the core 6 motif LGGHSL was found to be involved in the formation of the thioester(5, 20) . In order to assign the location of a putative thioester module, the ability of the constructed GrsA deletions in thioester formation was tested.

The results shown in Fig. 4clearly indicate that the deletions PheATS-His and PheAT-His restored the ability for thioester formation, whereas the deletion within GrsA mutant PheA-His caused complete loss of this activity. We found almost wild type levels for the first two constructs and decreased binding capacity of 13% for mutant PheA-His. This value remains in good correspondence with data obtained for TycA mutants, which are incapable of covalent binding of substrate amino acid. The latter deletion, PheA-His, which lost the ability to covalently bind phenylalanine, is devoid of the putative thioester binding site located within core 6 (Fig. 1, b and c)(5, 20) . Therefore, the results obtained represent additional support for the possible involvement of the deleted region in thioester formation. In this context it is important to notice that the 556-aa residue fragment PheA-His is still active in amino acid recognition and ATP-dependent activation. The C-terminal 100 aa of PheAT-His including core 6 may represent the site of substrate covalent binding.


Figure 4: Thioester formation of GrsA and derivatives. The wild type protein and the GrsA derivatives were charged with [^14C]phenylalanine in the presence and absence of ATP. The vertical bars represent the amount of covalently bound [^14C]phenylalanine (in fmol) per pmol of enzyme.



Cleavage of the covalently bound [^14C]Phe from GrsA and derivatives was performed in 80% performic acid and 6% hydrogen peroxide, indicating the thioester nature of this linkage(35) . To determine the racemization capacity of the GrsA deletions, the protein-[^14C]Phe complexes were cleaved and subjected to chiral plate TLC. The D- and the L-forms of the cleaved phenylalanine were distinguished by their R(F) values of 0.49 and 0.59 in solvent A, respectively(20) . Fig. 5shows the autoradiography and the radioactivity scan of the chiral high performance thin layer chromatography plate. The wild type GrsA protein revealed racemase activity as determined by signals for the D- and L-enantiomer, respectively (Fig. 5, lane 2). The C-terminal 291-aa deletion of PheATS-His (Fig. 5, lane 3), which is devoid of a putative racemase module, shows only L-Phe and no D-isomer, indicating for the first time by biochemical studies a possible role of this C-terminally located region in racemization of the covalently bound amino acid.


Figure 5: Analysis of the protein-[^14C]phenylalanine cleavage products by chiral plate TLC. a, autoradiography of separated cleavage products. Commercially available L-[^14C]Phe, slightly contaminated with D-[^14C]Phe, was used as control (lane 1). Cleavage products (D-Phe, L-Phe, and N-formyl-Phe; (35) ) of the wild type protein (lane2) and the GrsA deletion mutant PheATS-His (lane3) are shown. b, radioactivity scan of lanes2 and 3.




DISCUSSION

The results presented define by deletion studies on a peptide synthetase, GrsA, for the first time the minimal size of an amino acid activating domain and determine the locations of integrated modules involved in ATP-dependent acyladenylation, thioester formation, and epimerization. We dissected the multifunctional peptide synthetase GrsA and cloned, overexpressed, and purified the truncated fragments. The successive deletion at the C terminus of GrsA resulted in the defined mutants PheATS-His, PheAT-His, and PheA-His, possessing molecular masses of 92.7, 76.2, and 64.3 kDa, respectively. The purified deletion mutants were examined for their amino acid-dependent ATP-PP(i) exchange activity and specificity, their substrate affinity, their capacity for covalent binding of phenylalanine, and their ability to racemize the cognate amino acid.

All three mutants restored full ATP-PP(i) exchange activity, revealing a core fragment of this activity consisting of 556 aa. This adenylate-forming area comprises the core sequences 1-5 and possesses nearly half the size of native GrsA (1098 aa residues). With the exception of core 1, these core motifs are believed to be involved in ATP binding and hydrolysis(20, 21, 22) . The extent of the present core motifs coincides with the conserved regions reported for a family of adenylate-forming enzymes, including EntE from E. coli, luciferase from P. pyralis, 4-coumarate:CoA ligase from rat, and acetyl:CoA synthetase from A. nidulans(2, 7) . Interestingly, the substrates of these enzymes are entirely carboxyl acid derivatives; however, even the 64.3-kDa core fragment (PheA-His) only activates phenylalanine but not the phenylalanine carboxyl acid derivative 3-phenylpropionic acid. These findings and the exclusive activation of the cognate amino acid strongly suggest the location of the amino acid recognition site within the first 556 aa of native GrsA enzyme. We also investigated the binding affinities between truncated GrsA deletions and the substrates ATP and Phe, respectively. The Michaelis-Menten constants K(m) determined revealed only slight alteration in the binding affinities and corresponded to values published for several other native peptide synthetases(20, 38) .

In conclusion, all these findings indicate an almost undisturbed structure/function relationship of the truncated GrsA derivatives even in the case of a C-terminal 542-aa deletion from the native peptide synthetase (PheA-His). For the first time, a 556-aa fragment was identified by biochemical studies, which mediates amino acid recognition and ATP-dependent activation upon a peptide synthetase. Therefore, it is designated acyladenylation module. It comprises the first five core motifs and possesses nearly half the size of native GrsA (Fig. 1c).

To study the formation of the GrsA phenylalanine thioester linkage, deletion mutants were tested for amino acid incorporation. We found, in good correspondence to earlier investigations, that the covalent binding of the substrate amino acid to recombinant GrsA and derivatives expressed in E. coli is very inefficient(20) . Because the Phe-activating domain seems to be a poor substrate for the holo-acyl carrier protein synthetase, which normally catalyzes the transfer of the cofactor 4`-phosphopantetheine from coenzyme A to the acyl carrier protein(39) , only about 14% of recombinant GrsA from E. coli could be charged with [^14C]Phe. A similar finding was also observed for the peptide synthetase TycA, when expressed in E. coli(20) .

Sequence analysis revealed homologous domains for a diverse number of multifunctional peptide synthetases(7) . The conserved domains of these enzymes bear, in addition to a sole adenylation domain (see above), an extension of about 100 aa at their C terminus. This additional region comprises the putative 4`-phosphopantetheine binding motif LGGHSL(5, 20) . We imitated both forms of adenylate-forming enzymes by the constructed deletion mutants PheA-His and PheAT-His, and we studied their ability for covalent binding of the cognate amino acid. The results shown verify that the deletions PheATS-His and PheAT-His almost restored the wild type level for covalent binding of the substrate amino acid, whereas the deletion of the putative thioester binding fragment within the GrsA derivative PheA-His led to complete loss of thioester formation ability. Therefore, the findings represent additional support for the role of the deleted region as a thioester formation module. The state of this C-terminal area as a functional unit was verified by the identification of a second class of domain (type II) found in fungal peptide synthetases. These type II domains carry between core 5 and 6, in addition to type I domains, an insertion of about 430 aa residues that may function as a N-methyltransferase. All type II domains known, one in the enniatin synthetase and seven within the deduced amino acid sequence of the 1,689-kDa cyclosporin synthetase, correspond with the occurrence of N-methylated amino acids in the peptide antibiotics(12, 13) . This observation demonstrates that the acyladenylation module and the thioester-forming module could be separated by an additional unit necessary for the modification (N-methylation) of the activated amino acid.

Another well known function of the native peptide synthetase GrsA is the racemization of the substrate amino acid, which takes place on the thioesterified phenylalanine(39) . A comparison of GrsA with other peptide synthetases capable of racemizing the L-configurated substrate amino acid revealed four homologous motifs at the C terminus, which are absent in the non-racemizing domains. The deletion of this putative racemization site, which is located within the C-terminal 291 aa residues of GrsA, was part of the mutant PheATS-His. This GrsA derivative is able to activate phenylalanine as acyladenylate and to form a covalently bound thioester, but completely loses the racemization activity observed in the wild type protein. This finding indicates the possible involvement of the C-terminal region of GrsA in racemization of the substrate amino acid.

In conclusion, our dissection studies on the multifunctional GrsA protein as a model for a type I domain of peptide synthetases clearly point out for the first time the locations of modules involved in substrate recognition, acylation, thioester formation, and racemization. Based on biochemical data we identified the amino acid-activating domain of a peptide synthetase as the 656-aa region, which is conserved within a superfamily of adenylate- and thioester-forming enzymes. The extent of those domains represent nearly half the size of any proteolytic fragments so far described for specific amino acid activation(10, 14, 15, 16, 17) . Therefore, this finding will be very helpful for the development and realization of domain exchanging experiments in peptide synthetases.


FOOTNOTES

*
This work was supported by the Deutsche Forschungsgemeinschaft and by the Fond der chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-6421-285722; Fax: 49-6421-282191; marahiel{at}ps1515.chemie.uni-marburg.de.

(^1)
The abbreviations used are: aa, amino acid(s); PCR, polymerase chain reaction; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; PAGE, polyacrylamide gel electrophoresis; PheA, PheAT, and PheATS, deletion mutants of the phenylalanine (Phe)-activating GrsA protein containing adenylation (A), thioester formation (T), and spacer (S) modules, respectively.


ACKNOWLEDGEMENTS

We thank Inge Schüler for technical assistance and Stefan Borchert, Kürsad Turgay, Martin Gocht, and Oliver de Peyer for discussion and comments on the manuscript.


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