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1 Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA
2 Beijing Tuberculosis and Thoracic Tumor Institute, Beijing 101149, China
3 Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, Technologiepark 927, B-9052 Ghent, Belgium
4 University of Georgia, Complex Carbohydrate Research Center, 315 Riverbend Road, Athens, GA 30602, USA
Correspondence
Michael R. McNeil
mmcneil{at}colostate.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-D-ribose-1-diphosphate : decaprenyl-phosphate 5-phosphoribosyltransferase (DPPR synthase). The enzyme is essential for growth and thereby an important target candidate for the development of new tuberculosis drugs. Although membrane proteins are an important subset of targets for current antibacterial agents, details about the structures and the active sites of such proteins are often not readily available by X-ray crystallography. To begin a different approach to the issue, homologues from Mycobacterium smegmatis and Corynebacterium glutamicum were expressed in Escherichia coli and shown to be active DPPR synthases. This was followed by bioinformatic analyses of the aligned sequences and then by site-directed mutagenesis of amino acids identified as likely to be important for activity. The results suggested that the enzymic synthesis of decaprenyl-phosphate 5-phosphoribose (DPPR) occurs on the cytoplasmic side of the plasma membrane. Amino acid substitutions showed that the predicted cytoplasmic N-terminal region and two cytoplasmic loops are involved in substrate binding and/or catalysis along with parts of some adjoining inner membrane regions. The enzyme lacks the classical phosphoribose diphosphate (pRpp) binding site found in nucleic acid precursor enzymes of both prokaryotes and eukaryotes but instead contains a conserved NDxxD motif required for enzymic activity. Thus, it is plausible that this DPPR synthase has a pRpp binding site that is different from that of the classical eukaryotic enzymes, and further work to develop inhibitors against this enzyme is thereby encouraged.
Present address: TB Research Group, Veterinary Laboratories Agency, Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Xin et al., 1997; Scherman et al., 1996, 1995). For that reason the biosynthesis of DPA has been studied (Huang et al., 2005; Mikusova et al., 2005; Xin et al., 1997; Scherman et al., 1996), yielding the information that it is synthesized in four steps (Mikusova et al., 2005). The first step of the synthesis is the formation of decaprenylphosphoryl-β-D-5-phosphoribose (DPPR) from decaprenyl phosphate (DP) and phosphoribose diphosphate (pRpp) (Fig. 1). The enzyme responsible for this step, 5-phospho--D-ribose-1-diphosphate : decaprenyl-phosphate 5-phosphoribosyltransferase (DPPR synthase), in Mycobacterium tuberculosis has recently been cloned and identified (Huang et al., 2005). This enzyme has the potential to be a good drug target since it catalyses a reaction not found in humans. In this study the amino acid sequence of DPPR synthases from several species of Actinomycetales and also from an Azorhizobium strain were compared and their membrane-spanning domains were predicted. Site-directed mutagenesis of amino acids hypothesized to be involved in substrate binding and/or catalysis was then performed. The results led to the identification of specific domains and amino acids required for substrate binding and catalytic activity.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
Cloning and expression of genes encoding DPPR synthase.
To search for homologues of the DPPR synthase of M. tuberculosis, the amino acid sequence of Rv3806c was used as a query in a homology search of the TIGR database (http://tigrblast.tigr.org/cmr-blast/). The forward primers used to amplify the DPPR synthase genes of M. tuberculosis (Rv3806c), Mycobacterium smegmatis (MSMEG_6401), Corynebacterium glutamicum (cg3189) and Azorhizobium caulinodans (noeC) (Mergaert et al., 1996) included an NdeI restriction site (underlined) while the reverse primers had a BamHI restriction site (underlined) downstream of the stop codon. The primer sequences were: M. tuberculosis (Fw) 5'-CATATGAGTGAAGATGTGGTGACTCAACC-3', (Rev) 5'-GGATCCCTAGCCGAAGGCAACAGCGGC-3'; M. smegmatis (Fw) 5'-CATATGGATGCCACCCACATGAGTG-3', (Rev) 5'-GGATCCTCAGCTGAAATAGATGGCCGC-3'; C. glutamicum (Fw) 5'-CATATGAGCGAACACGCCGCTGAAC-3', (Rev) 5'-GGATCCTCAAAACATCGGCATGATGTAC-3'; A. caulinodans (Fw) 5'-CATATGTGGAATAAGGAATGGCCCG-3', (Rev) 5'-GGATCCTCAACCAATCGATTGCGACCG-3'.
Vent DNA polymerase (New England Biolabs) was used in the PCR reactions. Purified DNA fragments were cloned into pSTBlue-1 plasmid (blunt vector) using the Perfectly Blunt cloning kit (Novagen). DH5 competent cells (Invitrogen) were then transformed with 1 µl of the ligation mixture. Single colonies were isolated and plasmids carrying the correct insert were identified by DNA sequencing. The genes were then cloned into pET16b, in-frame with the coding sequence of the histidine tag at the 5' end, by using the restriction sites NdeI and BamHI. The resulting expression vectors were transformed into Escherichia coli ER2566 (New England Biolabs) and protein expression was induced by the addition of 0.4 mM IPTG followed by incubation at room temperature for 4 h. The cells were harvested by centrifugation and resuspended in buffer A [50 mM MOPS (pH 8.0), 10 mM MgCl2, 5 mM β-mercaptoethanol]. The cell suspension was disrupted either by probe sonication on ice with a Sanyo Soniprep 150 (10 cycles of 60 s on and 90 s off) or by French pressure cell FC020K (Cell Scientific) (two presses at 12 600 p.s.i.=8687 kPa). The suspension was centrifuged at 20 000 g for 30 min, to generate supernatant and pellet. The supernatant was then ultracentrifuged at 100 000 g for 1 h, and the resulting membrane-enriched pellet was removed and homogenized in a small volume of buffer A to a concentration of approximately 2 mg protein ml–1, as determined by Coomassie Plus Protein Assay reagent (Pierce).
Site-directed mutagenesis.
Mutagenesis was accomplished by overlapping extension as described by Ho et al. (1989), using two internal overlapping primers (one forward and one reverse with the same desired mutation) in combination with forward and reverse primers (as above). The resulting PCR products, with specific pre-determined mutations, were cloned into the plasmid pSTBlue-1 using the Perfectly Blunt cloning kit, and the presence of the correct mutations was confirmed by sequencing. Subsequently, the genes modified by site-directed mutagenesis were cloned into pET16b and transformed into and expressed in E. coli ER2566, as described above.
SDS-PAGE and immunodetection.
Purified cell membranes were incubated with loading buffer for 5 min at 45 °C (rather than boiled), as is required to visualize the DPPR synthase protein (Huang et al., 2005). Proteins were separated by 12 % SDS-PAGE and visualized by using GelCode Blue Stain reagent (Pierce). For immunodetection, the proteins were transferred to PVDF membranes, and monoclonal anti-polyhistidine (mouse IgG2a isotype) and conjugated anti-mouse-IgG-alkaline phosphatase were used as primary and secondary antibodies, respectively.
DPPR synthase assay.
The radiolabelled substrate, phospho[14C]ribosyl diphosphate (p[14C]Rpp), was prepared enzymically from [14C]glucose (American Radiolabelled Chemicals) as described by Scherman et al. (1996). DP (Indofine Chemical) was dissolved in 7.5 % (w/v) CHAPS to a concentration of 1 mg DP ml–1. M. smegmatis mc2155 membranes (Scherman et al., 1996) were used as the positive control and E. coli membranes purified from ER2566, which harboured empty pET16 vector, were used as the negative control. A standard reaction mixture of 30 µl contained 0.18 nmol [45 nCi (1665 Bq)] HPLC-purified p[14C]Rpp, 4 µg DP, 5 µg membrane protein and 0.25 % (w/v) CHAPS in buffer A. After a 15 min incubation at 37 °C, the enzymic reaction was stopped by adding chloroform and methanol to give a final chloroform/methanol/water ratio of 8 : 4 : 3 (by vol.). After centrifugation to generate two phases, an aliquot of the lower organic layer was then dried and radioactivity was measured on a Trilux MicroBeta scintillation counter (Perkin-Elmer/Wallac). Periodically, to confirm the identity of the product, TLC analysis was performed. In this case, the remaining organic phase was evaporated to dryness and the pellet was resuspended in chloroform/methanol/1 M ammonium hydroxide [65 : 25 : 4 (by vol.)]. Samples were spotted on a Silica gel 60 F254 TLC plate (Merck) and developed in chloroform/methanol/water/concentrated ammonium hydroxide/1 M ammonium acetate (180 : 140 : 23 : 9 : 9, by vol.). Autoradiography was carried out by exposing the TLC plate to X-OMAT AR film (Kodak) for 2 days at –80 °C. For the determination of Km values for pRpp and DP, the reaction time was reduced to 5 min and various concentrations of non-radioactive pRpp and/or DP were used. After separation of the organic layer from the aqueous layer by centrifugation, the radioactivity in a known volume of the organic phase was determined by liquid scintillation counting (as above). The data were then analysed by non-linear regression using the program Grafit 5.0 (Leatherbarrow, 2001).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) hit one strong homologue in each of the complete sequenced genomes of mycobacteria, corynebacteria and nocardiae (data not shown). This was the expected result due to the presence of arabinose in their cell walls (Dover et al., 2004). A BLAST search against the human database at NCBI showed the absence of any significant homologue to Rv3806c. The DPPR synthase candidates from M. smegmatis, C. glutamicum and A. caulinodans were selected, cloned and expressed in E. coli, essentially as done previously for DPPR synthase from M. tuberculosis (Huang et al., 2005). Western blot analysis using anti-His6-antibody showed that all these orthologues were expressed in the plasma membrane fraction (data not shown). The DPPR synthases of M. smegmatis and C. glutamicum were also shown to be capable of converting pRpp and DP to DPPR, as shown by a radioactive assay (Fig. 2a) and by confirmation of the identity of the products by TLC (Fig. 2b). However, the putative DPPR synthase from A. caulinodans, with 22 % amino acid identity to the M. tuberculosis homologue, showed no enzymic activity (Fig. 2a) despite being successfully expressed (see Discussion).
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. In addition, the membrane-spanning regions for each of these proteins were predicted by Phobius (Kall et al., 2004) and are included in the figure. The topology pattern was very similar among the four proteins, but the first transmembrane (TM) domain of NoeC was not predicted. These proteins are obviously integral membrane proteins, largely embedded in the plasma membrane, but they carry several larger soluble domains i.e. the N terminus and three cytoplasmic loops (CL-I, CL-II, CL-IV and CL-V in Fig. 3). No regions other than small connecting peptides were predicted to be located on the extracellular side of the membrane. Among the four aligned homologues in Fig. 3, 54 invariant amino acids were found and they tended to cluster in three regions. These regions are the cytoplasmic N terminus and the beginning of TM domain 1 (TM-1), several residues of TM-2 and the subsequent loop CL-II, and parts of TM-6 and CL-IV. The last approximately 50–70 amino acids in the C-terminal region, including CL-V, showed lower sequence conservation than the other regions of these four proteins.
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). This motif, in closely related forms (where N can be substituted for D or E), is found in UbiA phenyltransferases (Hemmi et al., 2004) and E-prenyl diphosphate synthases (Wang & Ohnuma, 1999). It is also found in prenyl diphosphate transferases (Brauer et al., 2004; Saiki et al., 1993) such as CyoE, which prenylates haem O (Saiki et al., 1993). Finally it is found in phosphoribosyl transferases (Craig & Eakin, 2000). In all these instances a pyrophosphate group is present on one of the substrates. Indeed, in the E-prenyl diphosphate synthases two of these motifs are present (Wang & Ohnuma, 1999), presumably one for each diphosphate substrate (isopentyl diphosphate and the growing allylic diphosphate). In the case of the substrates for DPPR synthase, the diphosphate-leaving group is part of pRpp while the DP isoprene is present as a monophosphate rather than diphosphate. Thus, the NDxxD motif is most likely to be important for pRpp binding and subsequent dissociation of the PPi instead of (or perhaps in addition to) DP binding.
We also searched the sequences of the DPPR synthases for homology to pRpp and DP binding sites that have been described in enzymes known to use these substrates. However, using the conserved pRpp binding motif of pyrR of M. tuberculosis (Kantardjieff et al., 2005), no corresponding motif was found among the DPPR synthase homologues. In a search for a binding region for DP, three polyisoprene-recognition sequences from NeuE, Dpm1 and Alg7 (Zhou & Troy, 2005) were aligned with the DPPR synthases. Interestingly, a region of Alg7 (L81FVMFIYIPFIFY), weakly aligned with residues 58–70 of M. tuberculosis DPPR synthase, with the first two phenylalanine residues (F59 and F62) and the second tyrosine residue (Y70) being present in both Alg7 and all four sequences studied here (Fig. 3). This sequence analysis of the DPPR synthase homologues gave us clues about where functional sites may occur, since amino acid residues/motifs directly involved in specific functions are most often well conserved.
Site-directed mutations in M. tuberculosis DPPR synthase
Invariant amino acids can be found along nearly the entire alignment (Fig. 3), but the three main clusters of conserved residues (in CL-I, CL-II and CL-IV), including the NDxxD motif discussed above, stand out. The topology prediction suggested that these clusters are predominantly found in the cytoplasmic domains or as part of the neighbouring TM domains, and led us to believe that many of these conserved residues may form functional site(s) in the native structure of the protein. Thus, selected amino acids from these three clusters were changed by site-directed mutagenesis in the M. tuberculosis DPPR synthase and the enzymic activity for each point-mutated version was investigated (see Methods). The results are shown in Table 1. In all cases but two (D77A and A66F) the mutated protein was expressed in the membrane fraction, in a fashion indistinguishable from that of the wild-type enzyme, as shown in Fig. 4.
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It is reasonable to suggest that the TM region upstream of the NDxxD motif is part of a pocket for the lipid substrate DP. Point mutations in that highly conserved region would, in that case, affect the enzymic activity and binding of DP. Mutations F59A and F62A had only small effects on the activity, while Y70A led to a complete loss of activity, suggesting a critical role for this aromatic amino acid predicted to be located at the plasma membrane interface. The conversion A66F resulted in a protein that was not expressed in the membrane (Table 1).
The final conserved region among the DPPR synthases is found in domain TM-6 and the subsequent CL-IV (Fig. 3). It is of interest to note that the proteins of the MenA family (enzymes which add a polyisoprene unit to dihydroxynaphthoic acid) have a similar predicted membrane topology to that proposed here for the DPPR synthases, including a loop corresponding to CL-IV (Fig. 3). In the MenA family, this loop contains many conserved residues (alignment not shown). However, the sequence homology between CL-IV of the MenA family and the DPPR synthases is very low, suggesting a similar overall structure of the two protein families (DPPR synthase and MenA) but with diverse structural features in this loop due to recognition of different substrates. To determine the relevance of this loop in the DPPR synthases, three conserved residues in loop IV were changed (R192A, E195A and R201A). All three substitutions had a profound effect, with R192A and R201A more or less abolishing the activity, while E195A substantially reduced the activity. Such strong effects on activity from all three point mutations suggest that this loop makes a large contribution to the structure of a binding/active site.
Kinetic analysis of mutants
Kinetic analysis of the DPPR synthase was difficult due to the instability of pRpp and the low solubility of DP. Multiple analyses of the wild-type enzyme gave the Km values reported in Table 2, albeit with relatively large standard deviations. Hence subtle changes in Km were difficult to observe for mutants with the various amino acid changes. However, for several of the mutants, significant changes in Km were seen, as shown in Table 2. The exchange N29A gave a fourfold increase in Km for pRpp while the Km value for DP remained unaffected. Similarly, the mutation N73Q had a larger effect on the Km value for pRpp than on that for DP. In the case of substitutions D77E and E195A, the effects were associated with both the substrates, as indicated by strong increase in Km values for both pRpp and DP. None of the mutations that were part of the kinetic study had an effect on the Km for DP without having an effect on the Km for pRpp.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), in fact show DPPR synthase activity. Surprisingly, NoeC of A. caulinodans was inactive under our assay conditions, even though the protein was expressed and detected in the E. coli membrane fraction. This was especially unexpected since the sequence of NoeC was initially used to identify the DPPR synthase gene in M. tuberculosis (Huang et al. 2005). The inactivity of the recombinant protein is supported by the fact that we have failed in efforts to show DPPR synthase activity in crude A. caulinodans membranes (data not presented). It is possible that A. caulinodans utilizes a slightly different substrate, such as 5-phosphoribose 1-phosphate or ribose 1-diphosphate instead of pRpp. Alternatively, the lack of enzymic activity of NoeC may reflect improper folding or insertion into the membrane. A region perhaps especially liable to not being inserted properly into the membrane is the first TM domain of the A. caulinodans homologue. Indeed in our computer topology study, this transmembrane region was not even predicted. We do think that this transmembrane region is very likely to occur in the natural context of A. caulinodans; otherwise the whole topology of the enzyme would be different and specific regions that we have now shown to be required for activity would be on different sides of the membrane. Hence it is a real possibility that the first TM domain does not insert properly into the membrane when the A. caulinodans protein is expressed in E. coli. Ultimately, further experimentation will be required to resolve the reason for the lack of activity of the A. caulinodans homologue.
Identification of amino acid residues required for substrate binding and/or catalysis
The two substrates of the DPPR synthase, the sugar donor pRpp and the acceptor lipid DP, are components of the cytosol and the plasma membrane, respectively. It is therefore logical to suggest that the catalytic reaction forming DPPR (Fig. 1) occurs at the membrane interface, and towards the cytosol. Here the topology prediction and the sequence analysis of the DPPR synthases both pointed in the same direction, with a polarization of conserved residues in loops facing the cytoplasm. Many of these residues are likely to be involved in the function of the enzyme. In addition, the results of the site-directed mutagenesis presented here strongly confirm this. As summarized in Table 1, we clearly have identified residues involved in substrate binding and/or catalysis. Not surprisingly, all conserved residues in the NDxxD motif were essential for activity, as were the neighbouring residues Y70 and D81, suggesting that CL-II carries key structural elements for the activity of this enzyme. This is also the case for CL-IV, since amino acid substitutions R192 and R201 both essentially abolished the activity of the enzyme. This suggests that amino acids from CL-II and CL-IV are likely to form the major structures of the active site. The predicted TM-2 and TM-6 domains are directly related to these loops and they carry several conserved residues. In this regard it seems possible that the polyisoprene chain of DP interacts with either or both of these TM domains. However, the amino acid substitutions F59A and F62A made in TM-2 failed to significantly affect the activity of the enzyme. This suggests that the two phenylalanines exchanged here are not involved in a critical hydrophobic interaction. The kinetic analysis (Table 2) showed that N29 is specific for pRpp binding as its Km increased dramatically when this residue was mutated to an alanine while the Km for DP was not affected. In contrast, the Km for pRpp and DP both increased in the mutants N73Q, D77E and E195A.
All proposed mechanisms for reactions related to that of DPPR synthase involve nucleophilic substitution (either SN1 or SN2). In both instances it is likely that both substrates need to be bound to the enzyme simultaneously. In the process of obtaining Km values we consistently noted that higher concentrations of pRpp inhibited the formation of DPPR, both for the wild-type enzyme and for all of the mutants except those with very high Km values for pRpp. A likely explanation for the pRpp inhibition is that DP must bind to the enzyme before pRpp. Therefore DP binding is inhibited at higher pRpp concentrations, where pRpp binds to the apoenzyme and thereby inhibits the binding of DP and catalytic activity.
In total, the data presented in this study revealed an active site with contribution from residues of the cytoplasmic N terminus, part of TM- 2, and CL-II and CL-IV (Fig. 5). The N-terminal region is clearly involved in pRpp binding, given the effect of N29A on its Km. The NDxxD motif in loop II is essential for activity and, by homology to analogous enzymes, is likely to be involved in stabilizing the loss of PPi from pRpp. The perturbation D77E had a profound effect on the Km values for both pRpp and DP, suggesting fundamental contributions to the active site. In a similar fashion, all three residues mutated in CL-IV had profound effects on the enzymic activity; kinetic studies on the E195A mutant protein revealed that this region, much like the NDxxD motif, is fundamentally involved in the active site, including substrate binding. These results are a step towards the design of effective inhibitors against this enzyme, which is a potential target for new tuberculosis drugs (Huang et al., 2005). The lack of a classical pRpp binding site, as found in eukaryotic nucleic acid monomer synthetic enzymes, is encouraging in this regard.
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ACKNOWLEDGEMENTS |
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Edited by: W. Bitter
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 25 September 2007;
revised 13 November 2007;
accepted 15 November 2007.
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