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Journal of Bacteriology, May 2003, p. 2848-2855, Vol. 185, No. 9
0021-9193/03/$08.00+0 DOI: 10.1128/JB.185.9.2848-2855.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Tom T. Caradoc-Davies, and Iain L. Lamont*
Department of Biochemistry, University of Otago, Dunedin, New Zealand
Received 21 October 2002/ Accepted 3 January 2003
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ABSTRACT |
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INTRODUCTION |
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Nonribosomal peptide assembly is catalyzed by large multimodular enzymes known as nonribosomal peptide synthetases (NRPSs), and each module governs insertion of a single amino acid in the peptide product (reviewed in references 9 and 20). Each NRPS module is comprised of several semiautonomous domains. Together, these domains provide active sites for recognition and activation of an amino acid substrate (adenylation or A domains) and subsequent incorporation of the adenylated substrate into a peptide product (thiolation or T domains and condensation or C domains). The modules may also contain domains that modify the substrate by N methylation or epimerization prior to incorporation or that contain a thioesterase motif for cleavage of a nascent peptide from the multimodular complex. NRPS modules act in a coordinated fashion to produce a peptide product. This process has been termed the multiple carrier model (39), and it consists of an integrated and stepwise series of amino- to carboxy-terminal transpeptidation reactions.
A domains, which catalyze the adenylation of cognate amino acid substrates, are central to nonribosomal peptide synthesis. Peptide synthetase A domains are typically about 550 amino acid residues long and have 30 to 60% sequence identity (37). An alignment of their sequences shows that there are 10 regions (A domain motif sequences) that are particularly highly conserved and that these regions are interspersed with regions with lower or no sequence similarity (44). Peptide synthetase A domains exhibit approximately 15 to 30% identity with members of the acyl-coenzyme A synthase and luciferase families of enzymes (44). Enzymes in these families also catalyze the adenylation of carboxy substrates and together with the NRPSs form a superfamily of adenylating enzymes. The structures of two enzymes belonging to this superfamily have been determined; these enzymes are firefly luciferase (7) and the A domain of the NRPS GrsA from Bacillus brevis (8). The overall topology of the GrsA A domain is very similar to that of luciferase, despite the fact that the two proteins exhibit only 16% identity at the primary sequence level. NRPS A domains exhibit much greater identity with one another, so it can reasonably be concluded that the GrsA structure represents a prototype for all peptide synthetase A domains (20). This is particularly likely to be true for the active sites, and almost all 10 of the highly conserved A domain motif sequences are clustered around the AMP and phenylalanine substrate-binding sites in the GrsA structure.
Stachelhaus et al. (38) and Challis et al. (5) created data banks of over 150 A domain sequences. Alignments of these sequences showed that other residues around the active site, distinct from the 10 conserved motif sequences, are highly conserved among A domains that recognize the same amino acid substrates. These residues are likely to be responsible for substrate recognition. These alignments were used to generate consensus codes, which allowed the substrate specificity of uncharacterized NRPS modules to be inferred from sequence data. Stachelhaus et al. (38) demonstrated the validity of this approach by targeted alteration of residues predicted to determine the substrate specificity of GrsA, showing that alteration of only two residues in the active site was sufficient to cause adenylation of L-leucine instead of D-phenylalanine. Subsequently, Eppelmann et al. precisely altered the substrate specificity of two surfactin synthetase modules by similar site-directed mutagenesis of substrate-determining residues (14). Challis et al. (5) took the prediction-based approach a step further by using the GrsA structural model to generate a two-dimensional representation of the amino acid-binding pocket. They then superimposed the side chains of the predicted substrate-coding residues over each of the corresponding GrsA side chains in the binding pocket to create a two-dimensional model of the binding pocket for each substrate examined. This allowed a more detailed structural analysis of the residues that are predicted to play key roles in substrate recognition.
The analysis of amino acid residues in the inferred substrate-binding sites of NRPS A domains has enhanced our understanding of these enzymes. The aim of the research described in this paper was to utilize and extend this approach, in conjunction with biochemical methods, in order to analyze the activity of an NRPS designated PvdD that is required for synthesis of the siderophore pyoverdine by Pseudomonas aeruginosa. P. aeruginosa is a ubiquitous human pathogen and causes a wide range of infections (22). Pyoverdines, which are mixed hydroxamate-catecholate-type siderophores, are required for the virulence of this organism (31, 41). The pyoverdines synthesized by all strains of P. aeruginosa have an identical dihydroxyquinoline group, and attached to this group is a strain-specific peptide that is comprised of seven or eight amino acid residues and includes several atypical amino acids (30). PyoverdinePAO, secreted by P. aeruginosa strain PAO1, contains an eight-member partially cyclic peptide (Fig. 1). The pvdD gene is required for synthesis of pyoverdine (28). Analysis of the pvdD sequence showed that PvdD has two NRPS modules that have 91% sequence identity and 95% sequence similarity in the C-A-T domain repeats. The sequence identity is particularly high for the two substrate-determining A domains, and 521 of 523 amino acid residues are identical when the modules are aligned with each other. Merriman et al. (28) proposed that PvdD governs the incorporation of the two consecutive L-threonine residues at the C terminus of the pyoverdine peptide (D-Ser-L-Arg-D-Ser-fOHOrn-L-Lys-fOHOrn-L-Thr-L-Thr, where fOHOrn is L-N5-formyl-N5-hydroxyornithine).
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MATERIALS AND METHODS |
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DNA methods. Chromosomal DNA was prepared from P. aeruginosa PAO1 by using the rapid genomic miniprep method (6). The first module of pvdD was amplified from this DNA by using the Expand High Fidelity PCR system (Roche Molecular Biochemicals) and the following primers: 5'-GGGGGAATTCGGTAGGTGCAAGCACTC-3' and 5'-GGGGGAGCTCAGGTAGCCAGCAACGAATG-3'. The restriction enzymes EcoRI and SstI (introduced sites are indicated by underlining in the primer sequences) were used to directionally clone the amplified module in frame into pPROEX1 by standard methods (33). The resulting plasmid (pPROEX1::mod1) was transformed into E. coli BL21.
Purification of His-PvdD. A 5-ml culture of BL21(pPROEX1::mod1) was grown overnight at 37°C in LB containing ampicillin (50 µg/ml). The overnight culture was used to inoculate 500 ml of LB containing ampicillin (50 µg/ml), glycine betaine (2.5 mM), and sorbitol (1 M). This culture was grown to an optical density at 600 nm (OD600) of 0.5 at 18°C (about 2 days), and then isopropyl-ß-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.6 mM and the bacteria were incubated at 18°C until the OD600 was 1.5 (one additional day). Cells were harvested by centrifugation (10,000 x g, 10 min), and the pellet was frozen at -70°C until it was required. For protein purification, the cells were resuspended in 10 ml of lysis buffer (50 mM Tris-HCl [pH 8.5], 10 mM ß-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride [added fresh]) and sonicated with an MSE sonicator (15 10-s bursts at 10-s intervals, until 80% cell lysis was obtained, as estimated by the change in OD600). Lysates were centrifuged at 10,000 x g for 30 min to remove cellular debris. To analyze the insoluble fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the pellets were resuspended to obtain approximately 10% (wt/vol) solutions in 20 mM Tris-HCl (pH 8.0). Soluble fractions were analyzed directly. The recombinant His-PvdD, which comprises the first module of PvdD fused to a hexahistidine affinity tag by a peptide linker, was purified from the soluble fraction by using Ni-nitrilotriacetic acid (NTA) resin in conjunction with the protocol described by Life Technologies. All column purification steps were performed at 4°C in a temperature-controlled environment. Contaminating proteins were removed as described below.
ATP-PPi exchange assays. ATP-PPi exchange assays were performed by using a modification of the protocol described by Lee and Lipmann (21). The reaction mixture was prepared by combining one part each of a 20 mM amino acid substrate solution, an ATP buffer stock solution (10 mM ATP, 50 mM MgCl2, 2.5 mM EDTA, 5 mM dithiothreitol, 100 mM triethanolamine buffer [pH 7.7], 25% [wt/vol] sucrose), a 0.4% (wt/vol) bovine serum albumin solution in 10 mM NaOH, and a 10 mM PPi solution (pH 7.7) containing 20 µCi of 32PPi (0.15%). The 32PPi was added immediately prior to use to avoid precipitation as Mg2P2O7. The exchange reaction was initiated by incubating 50 µl of a 50-µg/ml His-PvdD solution with 200 µl of reaction medium in a 1.5-ml microcentrifuge tube at 37°C and was allowed to proceed for 15 min. For the negative control 50 µl of a 50 mM Tris-HCl (pH 8.5) solution was added in place of His-PvdD. The reaction was stopped by adding 500 µl of an activated charcoal suspension (50 ml [wet volume] of acid-washed activated charcoal, 250 ml of 14% perchloric acid containing 400 mM PPi, and enough distilled H2O to bring the volume to 1 liter) to the microcentrifuge tube. Following mixing, the charcoal was collected by passing the mixture through a 3-ml syringe attached to a Millipore filter containing 0.45-µm-pore-size filter paper. While contained within the filter, the charcoal was washed three times with 3 ml of PPi (100 mM, adjusted to pH 8.0 with HCl) and two times with 3 ml of distilled water. The ATP was then eluted from the charcoal with two 3-ml washes with an elution solution containing methanol and NH3 (1:1, vol/vol) (43) into a scintillation vial containing 9 ml of water. Cerenkov counts were obtained directly with this vial by using an LKB Rackbeta liquid scintillation counter. The background count was considered to be the lowest data point recorded for the no-enzyme-no-substrate control; this base value was subtracted from all other values.
Protein modeling.
The sequence of the PvdD A domain was aligned with that of GrsA by using a gapped ClustalW alignment, and putative substrate pocket-lining residues of PvdD were identified by using the strategies of Stachelhaus et al. (38) and Challis et al. (5). Next, the PvdD primary sequence was threaded through the GrsA structural model (PDB ID Code 1AMU) by using the program Swiss PDB viewer Deep View such that the PvdD residues suggested by the alignment to correspond to equivalent residues in the GrsA primary sequence were overlaid onto the 
carbon positions of the corresponding GrsA residues. The model resulting from the gapped alignment of GrsA with PvdD contained regions in which residues were not matched with partners from the opposite sequence. These gaps and insertions mostly occurred in loop regions; none occurred within 18 Å of the active site, and they were removed from the PvdD model. This structural model was then loaded into XtalView (27), and the active site residues of the two structures were compared. The L-threonine substrate of PvdD was inserted into the new model in an orientation identical to that of the corresponding L-phenylalanine substrate of GrsA (8). Diagrams of these models were made by using the Raster3D interface of XtalView (29). Additional diagrams were produced by using PyMOL.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The first module of pvdD was amplified by PCR, and the product was cloned into pPROEX1. The resulting plasmid (pPROEX::mod1) was transformed into E. coli BL21. Addition of IPTG to a culture of these bacteria resulted in production of a protein estimated to have a molecular mass of 
114 kDa (data not shown); this compared well with the predicted mass of the recombinant His-tagged PvdD module (121 kDa). N-terminal protein sequencing of the first six residues of this protein gave the expected MGHHHH signal, confirming that the correct protein (His-PvdD) had been produced.
BL21(pPROEX::mod1) cells were induced to make His-PvdD, grown in LB for 3 h at 37°C, sonicated, and centrifuged. Virtually all of the His-PvdD protein was in the insoluble fraction (data not shown). Previous researchers had great difficulty obtaining biologically active peptide synthetase domains or modules from inclusion body aggregates, and only one group reported successful renaturation of a functional synthetase (16), so it was preferable to develop a procedure for expressing His-PvdD in a soluble form. Several methods have been described that increase the likelihood that recombinant proteins will be expressed in E. coli in a soluble and active form. Two of these methods are induction of expression at a low temperature (35) and growth in the presence of the osmoregulator glycine betaine with a high concentration of sorbitol in the growth medium (3). Cells were grown in LB with or without betaine and sorbitol, and His-PvdD production was induced over a range of temperatures. Growth of cells at 18°C in the presence of betaine and sorbitol were the only conditions that gave significant amounts of soluble His-PvdD (data not shown).
Soluble His-PvdD was purified by using a nickel-containing resin (Ni-NTA). Fractions containing His-PvdD were pooled and concentrated. There were three major contaminating species, which had molecular masses of approximately 69, 57, and 42 kDa (Fig. 2, lane 2). The pooled material was reapplied to a fresh Ni-NTA column, and this step significantly reduced the amounts of the two smaller contaminants but not the amount of the 69-kDa protein (Fig. 2, lane 3). N-terminal sequencing of this protein showed that the eight N-terminal residues were MGKIIGID, the same residues as those in the 70-kDa DnaK protein of E. coli. DnaK is a member of the Hsp70 family of chaperone proteins (reviewed in reference 26), is often copurified with recombinant proteins that are overexpressed in E. coli, and on occasion may interfere with the optimal folding of these proteins (36).
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Determination of the substrate specificity of PvdD by ATP-PPi exchange.
ATP-PPi exchange assays were performed by using the protocol of Lee and Lipmann (21). These assays exploit the equilibrium nature of the aminoacylation reaction, which can be summarized as follows: E + S + ATP 
[E-S-AMP] + PPi, where E is the A domain of an NRPS enzyme, S is a substrate activated by the A domain of the NRPS enzyme, and [E-S-AMP] is the enzyme-aminoacyl adenylate complex. This is an equilibrium reaction, so if radiolabeled pyrophosphate is present, then labeled phosphate groups are introduced into ATP, but only if a suitable amino acid substrate is present.
Exchange assays were performed with each of the amino acids present in pyoverdine (L-ornithine was used in place of L-N5-formyl-N5-hydroxyornithine, because the latter compound could not be obtained commercially). His-PvdD activated only L-threonine (Fig. 3), which is consistent with the proposal that PvdD incorporates L-threonine residues into pyoverdine. The specificity of this enzyme was examined by carrying out ATP-PPi exchange assays in the presence of amino acids that have structural similarities to L-threonine (L-serine, D-threonine, and L-allo-threonine). The results (Fig. 3) indicated that His-PvdD has a high level of specificity for L-threonine.
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-carboxyl and -amino substrate group binding, are essentially invariant in all peptide synthetases, suggesting that all A domains present their cognate substrate residues to a discriminating pocket region in a conserved fashion; and (iv) as determined by other researchers, substrate specificity is correlated with residues predicted to line the active sites (5, 14, 38), implying that the substrate-specifying pocket regions are structurally similar.
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-amino and -carboxyl groups. Seventeen of these residues are identical to the corresponding residues in GrsA, and one is similar (Ile to Val). Another two residues are predicted to interact with AMP through the main chain carbonyl groups, and the side chains do not contribute to substrate binding. The extremely high level of sequence identity between the active site of GrsA and the predicted active site of PvdD further supports the validity of the modeling approach used.
The PvdD model was then transferred into XtalView, and the L-phenylalanine substrate from the GrsA model was altered to L-threonine. The -amino and -carboxyl groups and the and ß carbons of the L-threonine substrate were maintained in locations identical to the locations of the equivalent groups and atoms of the L-phenylalanine substrate in the GrsA model (Fig. 5A). The L-threonine side chain was then rotated around the ß carbon to investigate possible interactions with the pocket-lining residues. Deviation from the GrsA configuration of pocket-lining side chains was minimized in order to maintain the integrity of the modeling approach. In the resulting model of PvdD, the locations of the side chain and ß carbons of each of the pocket-lining residues were identical to those of the equivalent residues in GrsA. The general orientation of the side chains was also maintained, with one significant exception. In the GrsA structure, C331 is directed out from the pocket, away from the phenylalanine substrate, but the corresponding residue in PvdD, H806, is likely to play an extremely important substrate-selecting role, forming a discriminating hydrogen bond with the threonine side chain hydroxyl (Fig. 5). Bringing the imidazole ring of this histidine residue into the active site by rotation around the -ß carbon bond (relative to GrsA) allows a nitrogen atom of the side chain ring to hydrogen bond with the side chain oxygen atom of the threonine, with an estimated bond distance of 2.7 Å. The resulting rotamer is well within the normal range for histidine residues (13), and adoption of this conformation is probably promoted and stabilized by the side chains of M797, W701, and V805, which are predicted to take up the space that all other common histidine rotamers could occupy.
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DISCUSSION |
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The copurification of DnaK may indicate that the His-PvdD protein was initially improperly folded, as one intracellular function of DnaK is the targeting of misfolded proteins for degradation (4). Consistent with this idea, ATP-PPi exchange activity was not detected with L-threonine as the substrate when an enzyme preparation that included DnaK was employed in assays (data not shown). Moreover, it may be that association with DnaK inhibited complete folding of His-PvdD. Incubation of the preparation with ATP removed the contaminating DnaK species and perhaps promoted correct folding of the His-PvdD protein in the process.
Many peptide synthetases have a high degree of substrate specificity with only a single known amino acid substrate, whereas others have a lower degree of substrate specificity and can adenylate (activate) more than one amino acid substrate at discernible levels (reviewed in references 24 and 45). His-PvdD (and, by extension, PvdD itself) has high substrate specificity for L-threonine compared to the other amino acids that were tested. It is possible that PvdD activates the other substrates that were investigated but at a level that was too low to detect in our assay system. For example, there may have been slight activity with L-allo-threonine (Fig. 3), but the value was within the margin of error for the ATP-PPi exchange assays. The L-threonine-activating NRPSs SyrB and SnbC also have very high specificities for L-threonine (15, 42). VibF from Vibrio cholerae is an NRPS that catalyzes adenylation of L-threonine and also adenylates L-serine and L-cysteine, but only at about 1/100 of the rate for L-threonine (25).
A three-dimensional structural modeling approach was developed to generate a model of the PvdD active site region and to investigate the basis of the high substrate specificity of PvdD. In the absence of a crystal structure for PvdD, this model allows some understanding of the structural basis of the action of this enzyme. The two-dimensional model of Challis et al. (5) proposes that N740 of PvdD is involved in a key interaction with the L-threonine side chain hydroxyl. The three-dimensional model (Fig. 5) argues against this, since N740 is almost certainly too distant to interact directly with the threonine substrate. Furthermore, a serine residue is present at the corresponding position in SyrB (Fig. 4). Instead, it appears that the H806 residue is the key stabilizing and selective residue for substrate binding in PvdD. The cysteine in the equivalent position in GrsA does not appear to play an important role in substrate selection by this enzyme, but the three-dimensional modeling approach indicates that residues in this position play a role in substrate recognition for some peptide synthetases. Given the highly conserved nature of the predicted substrate-determining residues of other L-threonine-activating peptide synthetases (Fig. 4) (5, 38), the PvdD model could provide an accurate representation of the active site region for most L-threonine-activating A domains. The only exception is VibF (25), which has a completely different configuration of predicted substrate-determining residues (MFVAGLIW, compared with FWNIGMVH for PvdD [Fig. 4]) and presumably employs an unrelated mechanism of substrate recognition.
The PvdD model was also used to investigate the high substrate specificity of PvdD. As threonine residues are asymmetrical, PvdD should bind different stereoisomers in quite different orientations, but discriminating features of the substrate-binding pocket should restrict the access of all isomers except L-threonine to the active site. Identification of potential substrate-determining residues is an important outcome of the modeling approach and provides a basis for site-directed mutagenesis studies. The modeling approach could also be applied to other peptide synthetase A domains and could, for example, provide supporting evidence for assignments of substrate specificity or assist in the development of inhibitor molecules targeted against peptide synthetases. For example, an inability to produce pyoverdine has been correlated with substantially diminished virulence in P. aeruginosa strains (31, 41), so the development of inhibitors of PvdD could have important medical implications.
In conclusion, the experimental data and modeling approaches described here provide complementary insights into the nature of substrate selection by PvdD. The possibility of carrying out rational modification of active sites of peptide synthetases to enable incorporation of different amino acids into peptides has been demonstrated for GrsA (38) and two surfactin synthetases (14). The approaches that we describe here may allow this approach to be extended to a wide range of peptide synthetases, thereby enabling generation of a wide range of modified peptide products.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Present address: Department of Microbiology and Immunology, Stanford University, Stanford, Calif.
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REFERENCES |
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