(Received for publication, September 16, 1994; and in revised form, December 28, 1994)
From the
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-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.
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) ()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.
The amplification of the grsA fragments was performed using Deep
Vent® DNA polymerase, 10
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
® 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 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`.
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.
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 (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.
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 exchange reaction of the GrsA derivatives. The ATP-PP
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.
All deletion mutants bind the substrates ATP and
phenylalanine with nearly the same affinity as the wild type protein.
The K 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
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.
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 [C]phenylalanine in the presence
and absence of ATP. The vertical bars represent the amount of
covalently bound [
C]phenylalanine (in fmol) per
pmol of enzyme.
Cleavage of the covalently bound
[C]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-[
C]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
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-[C]phenylalanine cleavage products by
chiral plate TLC. a, autoradiography of separated cleavage
products. Commercially available L-[
C]Phe, slightly contaminated with D-[
C]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.
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 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 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
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 [C]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.