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Journal of Bacteriology, September 2006, p. 6081-6091, Vol. 188, No. 17
0021-9193/06/$08.00+0     doi:10.1128/JB.00338-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The Structure of MbtI from Mycobacterium tuberculosis, the First Enzyme in the Biosynthesis of the Siderophore Mycobactin, Reveals It To Be a Salicylate Synthase

Anthony J. Harrison,^1,2 Minmin Yu,⁴ Therés Gårdenborg,^1,2, Martin Middleditch,² Rochelle J. Ramsay,^1,2 Edward N. Baker,^1,2,3 and J. Shaun Lott^1,2*

Centre for Molecular Biodiscovery,¹ School of Biological Sciences,² Department of Chemistry, University of Auckland, Auckland, New Zealand,³ Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720⁴

Received 9 March 2006/ Accepted 21 June 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
The ability to acquire iron from the extracellular environment is a key determinant of pathogenicity in mycobacteria. Mycobacterium tuberculosis acquires iron exclusively via the siderophore mycobactin T, the biosynthesis of which depends on the production of salicylate from chorismate. Salicylate production in other bacteria is either a two-step process involving an isochorismate synthase (chorismate isomerase) and a pyruvate lyase, as observed for Pseudomonas aeruginosa, or a single-step conversion catalyzed by a salicylate synthase, as with Yersinia enterocolitica. Here we present the structure of the enzyme MbtI (Rv2386c) from M. tuberculosis, solved by multiwavelength anomalous diffraction at a resolution of 1.8 Å, and biochemical evidence that it is the salicylate synthase necessary for mycobactin biosynthesis. The enzyme is critically dependent on Mg²⁺ for activity and produces salicylate via an isochorismate intermediate. MbtI is structurally similar to salicylate synthase (Irp9) from Y. enterocolitica and the large subunit of anthranilate synthase (TrpE) and shares the overall architecture of other chorismate-utilizing enzymes, such as the related aminodeoxychorismate synthase PabB. Like Irp9, but unlike TrpE or PabB, MbtI is neither regulated by nor structurally stabilized by bound tryptophan. The structure of MbtI is the starting point for the design of inhibitors of siderophore biosynthesis, which may make useful lead compounds for the production of new antituberculosis drugs, given the strong dependence of pathogenesis on iron acquisition in M. tuberculosis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mycobacterium tuberculosis, the cause of tuberculosis, is a devastating human pathogen, responsible for more than two million deaths annually (8). Although drugs that target actively growing M. tuberculosis are available, their effectiveness is compromised by the requirement for long treatment times (43) and the growing problem of multidrug resistance (15). A further problem is posed by the ability of the organism to enter a nonreplicating, persistent state after engulfment by activated macrophages in the lung (22). It is estimated that one-third of the world's population harbors a latent infection of this kind and is at risk of reactivation of disease (38).

Iron is essential for mycobacterial growth, as it is for virtually all living systems. For pathogenic bacteria, such as M. tuberculosis, iron acquisition is strongly correlated with virulence (45, 46). It has widely been assumed that macrophage-engulfed mycobacteria survive in an iron-poor environment within the phagosome, as has been demonstrated for Salmonella enterica serovar Typhimurium in epithelial cell vacuoles (19). However, recent studies have demonstrated that pathogenic mycobacteria (M. tuberculosis and Mycobacterium avium) actively increase the iron concentration in their phagosomes (53), presumably by capturing iron from endosomes containing transferrin (5), an ability not shared by nonpathogenic species, such as Mycobacterium smegmatis.

The physicochemical properties of free iron and its toxicity are such that sophisticated mechanisms have evolved to capture, solubilize, and assimilate this essential element. Mycobacteria, like most other microorganisms, synthesize chelating molecules called siderophores for this purpose (see references 10, 49, and 55 for recent reviews). Siderophores are produced in response to iron deficiency and are secreted into the environment, where they bind iron with high affinity and transfer it back into the cell (45). The siderophores produced by mycobacteria are of several types, notably, the salicylate-based mycobactins and the peptidohydroxamate-based exochelins. M. tuberculosis produces only mycobactin T (Fig. 1), although it is produced in several forms that differ in the nature of the acyl side chains attached to the central modified lysine residue. Both a water-soluble form, which is secreted into the external medium, and an insoluble, membrane-associated form are required in vivo for mycobacterial pathogenesis (13, 20, 45).

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FIG. 1. Chemical structure of mycobactin T, the siderophore of M. tuberculosis, annotated with the biosynthetic origins of its constituent components. In water-soluble variants, the acyl chain on the central modified lysine is 2 to 8 carbons long; in cell-wall associated variants, it can be up to 20 carbons in length. The drawing is based on data from reference 13.

The biosynthetic pathway by which mycobactin is produced has largely been elucidated (13, 45), although not all steps or enzymes have been specifically identified. Most of the enzymes that are implicated in the pathway are encoded by open reading frames that are clustered in the putative mbt operon, extending from Rv2377c to Rv2386c in the M. tuberculosis H37Rv genome (6). Deletion of mbtB (Rv2383c) showed that this gene, and by implication others involved in mycobactin biosynthesis, is essential for growth in macrophages and is required for virulence (12). This gene knockout also destroys the ability of the bacterium to accumulate iron when engulfed in the phagosome (53).

The last gene in the mbt cluster, Rv2386c, has also been shown, by genome-wide transposon mutagenesis (48), to be essential for the in vitro growth of M. tuberculosis. It was originally annotated as trpE2 (6) because of its similarity to trpE, the gene encoding the large subunit of anthranilate synthase, the enzyme that catalyzes the first committed step in tryptophan biosynthesis. However, like expression of other members of the mbt operon, the expression of Rv2386c is regulated by the iron response repressor IdeR. Under conditions of low iron, repression by IdeR is lost, and expression of Rv2386c and other mbt genes is induced (21, 47). The Rv2386c gene was thus reannotated as mbtI, putatively encoding the enzyme isochorismate synthase (44), which catalyzes the first step in the formation of salicylate and ultimately mycobactin. Sequence analysis of a number of chorismate-utilizing enzymes shows that Rv2386c (mbtI) clusters more closely with pchA, the gene encoding isochorismate synthase in Pseudomonas aeruginosa, than it does with trpE from a number of bacterial species (18). PchA has also been shown biochemically to act as an isochorismate synthase in the first step in the biosynthesis of the salicylate-containing siderophore pyochelin in P. aeruginosa (18).

Chorismate is a key intermediate in the biosynthesis of many essential aromatic compounds, being converted to prephenate in phenylalanine and tyrosine biosynthesis, anthranilate in tryptophan biosynthesis, p-aminobenzoic acid (PABA) in folate biosynthesis, p-hydroxybenzoate in ubiquinone and menaquinone biosynthesis, and salicylate in siderophore biosynthesis (54). The enzymes that catalyze these conversions of chorismate share a degree of sequence identity (typically on the order of 20% on a pairwise basis) that implies related structures. Three-dimensional structures are currently available for salicylate synthase (Irp9) from Yersinia enterocolitica (29), for anthranilate synthase (TrpE) from Sulfolobus solfataricus, Serratia marcescens, and Salmonella enterica serovar Typhimurium (31, 36, 50), and for aminodeoxychorismate synthase (PabB) from Escherichia coli (39). Anthranilate synthase is a hetero-oligomeric complex composed of the products of the trpE and trpG genes. TrpG is a glutamine amidotransferase which provides the amino group required in the biosynthesis of anthranilate. Aminodeoxychorismate synthase forms an analogous heterodimer, with PabA functioning as an amidotransferase. In contrast, the salicylate synthase from Y. enterocolitica is homodimeric. Although the structures of TrpE, PabB, and Irp9 share a common fold (29, 39), the fine structural differences that enable the production of different products by related enzymes (Fig. 2) are of great interest, particularly given the attractiveness of these enzymes for structure-based drug design.

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FIG. 2. Structurally characterized chorismate-utilizing pathways. Shown are the three analogous transformations of chorismate for which there is structural information about the enzymes involved: anthranilate synthesis, p-aminobenzoate synthesis, and salicylate synthesis. ADC, 4-amino4-deoxychorismate; ADIC, 2-amino-2-deoxyisochorismate.

Salicylate is a key compound required for the biosynthesis of siderophores in a number of bacterial species, and its production from chorismate is analogous to that of anthranilate in tryptophan biosynthesis or of PABA in folate biosynthesis (Fig. 2). In P. aeruginosa, a two-step conversion of chorismate to salicylate has been demonstrated, with the PchA protein acting as an isochorismate synthase (i.e., a chorismate isomerase, responsible for moving the hydroxyl group from position 4 to position 2 on the chorismate ring) and the PchB protein acting as an isochorismate-pyruvate lyase, cleaving the pyruvate moiety from the ring (17, 18). This is equivalent to the situation in PABA production in many bacteria, where the PabB protein facilitates chemical modification of the chorismate ring (in this case the addition of an amino group is provided by the amidotransferase PabA) but a separate protein, PabC, provides lyase activity (24, 37). In contrast, in tryptophan biosynthesis, the TrpE protein is able to act as both ring-modifying enzyme (again adding an amino group, in this case provided by TrpG) and lyase (1, 56). Recently, a TrpE-like salicylate synthase, Irp9, which is capable of both ring isomerization and pyruvate lyase activity, from Y. enterocolitica has been characterized (30, 41).

Here we describe the crystal structure of MbtI, the gene product of Rv2386c from M. tuberculosis, the presumed isochorismate synthase that catalyzes the first step in mycobactin biosynthesis. We present both structural and biochemical evidence to demonstrate that MbtI does not function as an isochorismate synthase like PchA but instead as a salicylate synthase like Irp9, without the need for the separate isochorismate-pyruvate lyase (PchB) required in Pseudomonas (17). The structure of MbtI shows that the catalytic apparatus it shares with the chorismate-utilizing enzymes TrpE and PabB is essentially unchanged but that substantial variation occurs in the N-terminal region of the protein. Distinct from TrpE and PabB, but in common with Irp9, MbtI is found not to contain a tryptophan binding site in its N-terminal region.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Protein expression, purification, and crystallization. Native MbtI was expressed and purified as described previously (26). Selenomethionine (SeMet)-substituted MbtI was prepared in a similar manner by expression of the construct in DL41(DE3)-CodonPlus-RP cells grown in LeMaster defined medium containing 25 µg/ml SeMet and appropriate antibiotics (14). Purification was carried out as for native protein, except that all buffers were supplemented with 5 mM mercaptoethanol. Crystallization was carried out as described previously (26). Needle-shaped crystals, usually obtained as bundles of rods fused at one end, grew in 4 to 5 days by vapor diffusion from drops made by mixing equal volumes of protein solution (10 mg ml^–1 in 20 mM HEPES, pH 8.0, 1% glycerol) and precipitant solution (15% polyethylene glycol 4000, 0.2 M imidazole-malate, pH 6.0). Crystals of the SeMet-substituted protein grew under similar conditions but required a higher protein concentration (20 mg ml^–1).

Data collection and processing. For data collection, crystals were soaked in a cryoprotectant comprising reservoir solution (15% polyethylene glycol 4000, 0.2 M imidazole-malate, pH 6.0) supplemented with 20% (vol/vol) glycerol and were then flash cooled at 113 K. Native data to 1.8-Å resolution were collected on beamline 14-4 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France, with an ADSC Quantum4 detector at a wavelength of 0.9393 Å. For the SeMet-substituted crystals, data were collected from a single crystal at two wavelengths (peak and inflection) on beamline 5.0.2 of the Advanced Light Source (Lawrence Berkeley Laboratory) by use of an ADSC Q315 charge-coupled-device detector. The data were indexed and integrated using MOSFLM (7). Both native and SeMet-substituted crystals proved to be orthorhombic, space group P2₁2₁2₁, with unit cell dimensions as follows: a = 51.82 Å, b = 163.36 Å, c = 194.93 Å (native) and a = 51.85 Å, b = 163.60 Å, c = 194.54 Å (SeMet). The native data set was 99.5% complete to 1.8-Å resolution, with an overall R_merge of 8.6% on intensities. Significant radiation decay occurred for the SeMet-substituted crystal, limiting data collection to two wavelengths. Full data collection statistics are given in Table 1.

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TABLE 1. Data collection and phasing statistics

Structure determination and refinement. Selenium positions were located and refined with autoSHARP (52) by using all data from 60 to 2.6 Å. All of the 24 expected selenium atoms in the asymmetric unit were located, confirming the presence of four molecules in the asymmetric unit. Real space density modification by solvent flattening and histogram matching was carried out using DM (9). Initial electron density maps were of sufficiently high quality to allow autobuilding using ARP/wARP (42). Initial autobuilding was able to place side chains for 362 residues out of a possible 450 from one molecule in the asymmetric unit. This model was then used for molecular replacement into the high-resolution native data set collected previously (26) by use of MOLREP (51). The resulting SigmaA-weighted electron density map showed clear density for many missing side chains and some loops. Several rounds of manual building using O (28) were then interspersed with refinement with CNS (2), using all data to 1.8 Å. Refinement strategy was judged by monitoring the free R factor (R_free) calculated from 5% of reflections. Initial simulated annealing was carried out with noncrystallographic symmetry restraints, bulk solvent correction, and an overall anisotropic B factor in place. In later stages, the noncrystallographic restraints were released, and individual, restrained atomic B factors were refined. Water molecules were added once the R factor dropped below 0.25. Glycerol, pyruvate, and imidazole molecules were modeled late in the refinement process, into peaks of >3 in F_o–F_c maps that were too large to be water. Analysis with PROCHECK (34) showed that 93.3% of residues were in the most favored region of the Ramachandran plot. Full details of the refined structure are shown in Table 2.

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TABLE 2. Refinement statistics

Enzyme assays. Salicylate production was monitored either by direct measurement using fluorescence (excitation wavelength, 305 nm; emission wavelength, 410 nm) or by reversed-phase high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Recombinant protein (10.2 µM) was incubated for 60 min at 30°C with 1.5 mM chorismate in 20 mM HEPES, 10 mM MgCl₂, pH 7.5, in a total volume of 1 ml. For HPLC-MS analysis, heptafluorobutyric acid (HFBA) was added to a final concentration of 0.005% (vol/vol). A sample (5 µl) was then injected immediately onto a Zorbax analytical 3.5-µm C₁₈ reversed-phase HPLC column (100 mm by 0.3 mm; 300-Å pore size). The flow rate was 6 µl/min. Solvent A was 0.1% (vol/vol) formic acid and 0.005% (vol/vol) HFBA in water, and solvent B was 0.1% (vol/vol) formic acid and 0.005% (vol/vol) HFBA in acetonitrile. The following gradient was used for all runs: 0 min, 2% solvent B; 20 min, 20% solvent B; and 28 min, 95% solvent B. The elution of compounds was monitored using a Q-STAR XL electrospray quadrupole mass spectrometer in negative ionization mode, using time-of-flight scanning over a range from 100 to 250 m/z.

To monitor salicylate production by MbtI using ¹H-nuclear magnetic resonance (NMR), the protein was buffer exchanged from 20 mM HEPES, 1% glycerol, pH 8.0, into 20 mM potassium phosphate buffer in D₂O, pH 7.0. Salicylate and chorismate standards (2 mM in water) were added to 20 mM phosphate buffer in D₂O immediately prior to use. The activity of the enzyme, as assessed by fluorimetry, was significantly reduced in phosphate buffer, and the reaction mixture (8.9 µM MbtI, 2 mM chorismate, 20 mM potassium phosphate buffer, 10 mM MgCl₂, pH 7.0) was therefore incubated for 18 h at room temperature. ¹H-NMR spectra were collected at room temperature by use of a Bruker Avance 600-MHz spectrometer equipped with a cryoprobe.

Protein structure accession number. The Protein Data Bank (PDB) accession number for MbtI is 2G5F.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Crystal structure of MbtI. The crystal structure of MbtI was solved by the multiwavelength anomalous diffraction method using crystals of the SeMet-substituted protein and was then refined with native data at 1.8-Šresolution to a final R factor of 0.205 and free R of 0.240. The crystal asymmetric unit was found to contain four MbtI molecules. The crystal packing, however, suggests a monomeric enzyme, consistent with size exclusion chromatography data which suggest a monomer in solution (data not shown). In the crystal asymmetric unit, the largest buried surface between molecules is 1,200 Ų (600 Ų per monomer, or 3.4% of the monomer surface) between molecules A and B and 1,040 Ų between molecules A and D, as calculated using the protein-protein interaction server (http://www.biochem.ucl.ac.uk/bsm/PP/server).

The final model conforms well to standard geometry, with 93.3% of residues having their main chain torsion angles in the most favored regions of the Ramachandran plot, as defined by PROCHECK. Molecules A, C, and D each comprise 434 residues (residues 15 to 449), with only the N-terminal residues 1 to 14 and C-terminal residue 450 having no interpretable electron density. Molecule B is additionally lacking density for residues 38 to 41 and 76. Overall, the four protomers are very similar: molecules A and B superpose with an overall root mean square (RMS) difference in C positions of 0.59 Å, and molecules C and D superpose with an RMS difference of 0.48 Å. Molecules A and B are fractionally more similar to each other than they are to molecules C and D (RMS differences: A/C, 0.93 Å; A/D, 0.88 Å; B/C, 0.97 Å; B/D, 1.01 Å), but there are no significant structural changes between the four monomers apart from localized differences in the active site, as described in more detail below. Full details of the final model are given in Table 2.

Molecular structure. The polypeptide of 450 residues forms one large single domain, with a fold that is basically the same as that previously described for the chorismate-utilizing enzymes Irp9, TrpE, and PabB. This fold can be described in terms of two /ß subdomains, each comprising a large antiparallel ß-sheet with helices packed against it. The two ß-sheets, one of 10 strands (subdomain I) and the other of 11 strands (subdomain II), pack approximately orthogonally across each other in the center of the molecule (Fig. 3), burying hydrophobic side chains between them. Helices pack on the outside of the ß-sheets, with five helices packed against the subdomain I ß-sheet and six packed against the subdomain II ß-sheet.

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FIG. 3. Ribbon diagram of the monomer structure of MbtI, colored blue to red from the N to the C terminus, with secondary structure elements as defined by DSSP, and labeled to be consistent with the nomenclature used for TrpE (50). The active site cleft is highlighted with a cyan circle.

Outside the region where the two ß-sheets cross, a deep cleft is formed between regions of the two subdomains. Several adventitiously bound ligands are found in this cleft, which by analogy with the substrate-bound structures of Irp9 (29) and TrpE (50) is identified as the active site. Clear electron density found adjacent to Arg405, from the ß19-ß20 loop, is modeled as a molecule of pyruvate in molecules A and B. Less-well-defined electron density in the same general area of the structure in molecules C and D is interpreted as glycerol, as discussed in more detail below. A further glycerol molecule is found adjacent to His334 from the ß16-ß17 loop in each of the four MbtI molecules. On the external surface, and without any obvious functional significance, planar electron density is interpreted as an imidazole molecule hydrogen bonded to Glu307 of each molecule.

Comparison with related enzymes. A BLAST search of the current nonredundant sequence database with the MbtI sequence reveals many homologous sequences, reflecting the widespread occurrence of enzymes that act on chorismate in a number of metabolic pathways. The most similar homologs in sequence terms identified include anthranilate synthase (TrpE) from a number of bacterial species, a 2,3-dihydroxybenzoate-AMP ligase from Ralstonia solanacearum, a putative p-hydroxybenzoate synthase from Nocardia farcinica, a putative salicylate synthase from Yersinia pestis, and the salicylate synthase (Irp9) from Y. enterocolitica.

An exhaustive search of the PDB using the secondary structure matching program SSM (32) shows that the closest structural match to MbtI is Irp9, the salicylate synthase from Y. enterocolitica (29). For this enzyme, which shares 34% sequence identity with MbtI, 330 residues can be superimposed onto corresponding residues in MbtI with an RMS difference in C atomic positions of 1.3 Å after applying a cutoff of 4.0 Å. There are small differences in terms of insertions and deletions when comparing MbtI to Irp9, and several loops are in considerably altered conformation between the two structures, notably between ß4 and ß4b, between ß4b and 2, between 3 and ß6, and at the start of 4. A somewhat lower correspondence is found with the anthranilate synthase (TrpE) enzymes from S. marcescens (50) and S. enterica serovar Typhimurium (36), which share about 23% sequence identity with MbtI and for which approximately 320 residues can be superimposed onto MbtI with an RMS difference of 1.5 Å. A striking feature is that both MbtI and Irp9 show much greater correspondence with TrpE and PabB over their C-terminal portions. Thus, when MbtI and S. marcescens TrpE are superimposed, residues 187 to 446 of MbtI match residues 245 to 510 of TrpE, with insertions or deletions occurring at only three sites: MbtI has one additional residue in the ß10-5 loop, one fewer in the ß13-ß14 loop, and six fewer in the ß15-6 loop. In contrast, in the N-terminal portion, which includes the tryptophan regulatory site in TrpE, major deletions occur in MbtI between ß2 and ß3a (7 residues), ß4 and ß5 (19 residues), and ß8 and 3a (17 residues). This pattern of structural similarity is echoed in sequence similarity: the N-terminal portion of MbtI (residues 1 to 186) shows sequence identity with TrpE at only 9 positions (7.5%) after structural alignment, whereas the C-terminal portion (residues 187 to 449) shows identity at 69 positions (26.2%).

A similar relationship exists between MbtI and PabB, although the structural correspondence is less close: with a 4.0-Å cutoff, 304 residues match with an RMS difference in C positions of 1.8 Å. Again the C-terminal region (residues 187 to 446) matches best and there are much greater differences in the N-terminal regions. A structure-based multiple sequence alignment of MbtI, TrpE, PabB, and Irp9 is shown in Fig. 4.

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FIG. 4. Structure-based multiple sequence alignment of MbtI with related enzymes of known structure. Irp9, salicylate synthase from Y. enterocolitica (PDB accession no. 2FN0/2FN1); PabB, p-aminobenzoate synthase from E. coli (PDB accession no. 1K0E/1K0G); TrpE, anthranilate synthase from S. marcescens (PDB accession no. 1I7Q/1I7S). Structural alignment was made using sPDBv (25) and subsequently edited by hand. The figure was drawn using ESPript (23). Identical residues are shown in white type with a black background, and similar residues which are conserved in all four sequences are shown boxed. (Similar groups as defined by ESPript are as follows: HKR, polar positive; DE, polar negative; STNQ, polar neutral; AVLIM, nonpolar aliphatic; FYW, nonpolar aromatic; PG, structure breakers; and C, thiol.) Secondary structure elements for MbtI are shown above the alignment, labeled to be consistent with the nomenclature used for TrpE (31, 50).

Active site. The active site of MbtI, identified by comparison to the product-bound forms of Irp9 and S. marcescens TrpE, is situated in a deep cleft between structural elements from the C-terminal portion of the molecule. The cleft takes the form of a long groove about 12 Å in length, 10 Å deep, and 7 Å wide. One wall of this groove is formed by strand ß21 and the following C-terminal helix, 11 (from subdomain I), and the other by the ß16-ß17 loop, helix 7, and the ß15-6 loop (from subdomain II), while the ß19-ß20 and ß12-ß13 loops form the bottom of the cleft (Fig. 3 and 5).

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FIG. 5. (A) Comparison of the active site regions of MbtI and the ligand-bound conformation of TrpE, showing the shift in the ß14-ß17-ß16 sheet. MbtI is shown in green, and TrpE is shown in blue with side chains in yellow. The bound Mg2+ ion is shown as a yellow sphere. In all cases except where noted, molecule B from the MbtI asymmetric unit is shown. (B) Overlay of the MbtI, TrpE, and Irp9 active sites. MbtI is shown in green, TrpE in yellow, and Irp9 in blue. (C) Pyruvate binding in the active site of MbtI. A molecule of pyruvate is shown modeled into residual density from a SigmaA-weighted Fo–Fc electron density map, contoured at 3, in the active site of molecule B in the asymmetric unit. Side chains are shown in green for residues that interact with the pyruvate, and hydrogen bonds are shown as dashed lines. This arrangement is identical in molecule A of the asymmetric unit. The alternative conformation of Arg405 in molecules C and D in the asymmetric unit, where no bound pyruvate is observed, is shown in light blue, represented using the side chain from molecule C. (D) The tryptophan binding site of TrpE is overlaid on the equivalent region of MbtI. The hydrogen bonding network in MbtI is shown as black dashed lines. Molecules are colored as described for panel A.

In both TrpE and PabB, the ß16-ß17 loop (residues 387 to 401 in S. marcescens TrpE and 328 to 342 in PabB) forms a mobile element that can adopt a closed or open conformation, apparently depending on whether ligands are bound or not. In our MbtI structure, this loop (residues 323 to 337) is in the open state, in contrast to the equivalent loop (residues 310 to 324) in the salicylate- and pyruvate-bound Irp9 structure, where it is in the closed state (29). The open conformation is equivalent to that seen in the tryptophan-bound form of TrpE (50), and a movement of some 5 to 7 Å at the tip of the loop would be needed to give rise to the closed conformation found in the Irp9 structure and previously seen in the benzoate-bound form of TrpE (50). The movement of this loop is accompanied by a similar but slightly smaller displacement of the adjacent ß14-6 connection (Fig. 5A).

The residues that are implicated in substrate binding and catalysis in Irp9, TrpE, and PabB are highly conserved in MbtI (Fig. 5B). The residues that coordinate the essential Mg²⁺ ion in Irp9 and TrpE, either directly or indirectly, are all conserved: Glu294, Glu297, Glu431, and Glu434 in MbtI correspond to Glu281, Glu284, Glu417, and Glu420 in Irp9 and to Glu358, Glu361, Glu495, and Glu498 in TrpE. No bound Mg²⁺ is present in MbtI, however, and in its absence Glu297 is turned away to hydrogen bond to His204. The residues that bind the salicylate in Irp9, Thr258, and His321 and the benzoate moiety in TrpE, Thr329, and His398 are also present in MbtI as Thr271 and His334. However, His334 is on the mobile ß16-ß17 loop and Thr271 on the adjacent ß14 strand, so are both swung away from the active site in this "open" form; a ligand-binding role for these residues may be the major factor in loop closure. Adjacent to this part of the active site is Ala269, which is conserved as alanine in the Irp9 and TrpE enzymes but corresponds to Lys274 in PabB, where it has been proposed to discriminate between C-2 and C-4 substitution on chorismate substrates (39) and has recently been shown to be of critical importance for catalytic activity, as it forms a covalent intermediate during the reaction (3, 4, 27).

At the bottom of the MbtI active site is electron density for a bound ligand, which is modeled as pyruvate in molecules A and B. Less-well-defined electron density in a similar but not exactly equivalent position is modeled as glycerol in molecules C and D. The electron density, B factors on refinement, and differences in binding mode all support these assignments. The putative pyruvate molecules in the A and B active sites both make several specific interactions with surrounding side chains; the carboxylate group forms a doubly hydrogen-bonded salt bridge with Arg405 (O1-N1 and O2-N2) and additional hydrogen bonds with Tyr385 O and the peptide NH of Gly419, and the hydroxyl group forms a hydrogen bond with Lys438 (Fig. 5C). The refined B factors of the pyruvate molecules, 35.4 Ų in molecule A and 25.0 Ų in molecule B, are very close to those for surrounding parts of the protein structure. After superposition of the MbtI with the TrpE and Irp9 structures, the positions of the proposed pyruvate molecules are displaced by only 1.5 Å and 1.7 Å, respectively, while making essentially the same hydrogen bonds. (In TrpE and Irp9, the equivalent arginine hydrogen bonds to O2 and O3 of the pyruvate via N and N1.) In contrast, the putative glycerol molecules in molecules C and D are not specifically bound, with the ligand in molecule C hydrogen bonded only to water and that in molecule D to Thr441 O1 and water, and have relatively high B values after refinement—60.7 Ų and 47.9 Ų in molecules C and D, respectively. Arg405 adopts markedly different conformations in the presence and absence of bound pyruvate, in the latter case swinging away from the active site to make hydrogen bonds with Asn231 and the backbone oxygen of Thr441. However, the rest of the active site seems unchanged in the absence of pyruvate.

Catalytic activity of MbtI. The presence of adventitiously bound pyruvate in the structure indicated that MbtI may be functioning as a salicylate synthase, i.e., that it has both isochorismate synthase (chorismate isomerase) and isochorismate-pyruvate lyase activities. To test whether this was the case, the activity of the enzyme was monitored fluorometrically (Fig. 6A). When incubated with chorismate, the enzyme gave rise to a product with a fluorescent emission maximum at 410 nm, which is consistent with salicylate. The reaction was absolutely dependent on the presence of Mg²⁺ and appeared to be strongly influenced by choice of buffer, with phosphate showing a marked slowing of reaction progress compared to Tris or HEPES. To confirm the identity of the fluorescent product as salicylate, the products of the reaction were separated by reversed-phase HPLC, compared to standard compounds, and identified by electrospray mass spectrometry (Fig. 6B). Chorismate alone eluted with a retention time (t_R) of 6.7 min, and a salicylate standard eluted with a t_R of 13.2 min. The product of the MbtI reaction yielded a peak with a retention time of 13.5 min. Electrospray mass spectrometric analysis indicated that this peak was composed of a species with an m/z of 137.0, which is consistent with salicylate and identical to the m/z ratio obtained from a standard solution of salicylate. An additional peak was observed on the chromatogram at a t_R of 6.0 min, showing an in-source fragmentation pattern identical to that produced by chorismate, and hence was assumed to be isochorismate.

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FIG. 6. Enzymatic activity of MbtI. (A) MbtI activity was monitored using fluorometric detection at an emission wavelength of 410 nm in the presence (+) and absence (–) of Mg2+. (B) Extracted ion chromatograms of MbtI reaction products, sampled over an m/z range from 137.0 to 137.1, compared to chromatograms of chorismate and salicylate incubated under the same conditions without enzyme. Chromatograms are all normalized to the highest peak. Chorismate yields a fragment with an m/z of 137 due to in-source fragmentation and also yields other fragments at an m/z of 207 and an m/z of 225 (traces not shown for clarity). (C) 1H-NMR spectroscopic analysis of salicylate production by MbtI, compared to spectra of chorismate and salicylate alone.

In order to further analyze the reaction, ¹H-NMR spectroscopy was carried out. After incubation of MbtI with chorismate, peaks corresponding to salicylate with _H values around 7.65, 7.30, and 6.80 were readily apparent (Fig. 6C). Additionally, peaks consistent with the presence of isochorismate, with _H values of 6.68 and 6.06, were also present, indicating that isochorismate was likely being formed as an intermediate in the reaction, as has been shown previously for Irp9 (30).

Tryptophan binding site. Although MbtI matches TrpE and PabB very closely over its entire C-terminal region and has an active site extremely similar to that of Irp9, there are substantial differences to these two enzymes in their N-terminal regions. These have the effect that the binding site for tryptophan, which gives feedback inhibition of TrpE and stabilization of the PabB structure, is absent from both MbtI and Irp9. Several key substitutions in MbtI, coupled with the truncation and displacement of its ß2-ß3 loop, combine to abolish the tryptophan binding site (Fig. 5D). The deletion of 7 residues from the ß2-ß3 loop and its displacement by 4 to 5 Å relative to that in TrpE cause the C atom of Cys50 (equivalent to Ser40 in TrpE) to occupy the position filled by the tryptophan C atom in both TrpE and PabB. In Irp9, Arg37 is the equivalent residue of Cys50 in MbtI and blocks the binding site in a similar manner, hydrogen bonding to Tyr42 and Asp380. The position of the tryptophan indole ring in TrpE and PabB is filled by the side chain of Tyr48 in MbtI (Tyr35 in Irp9), replacing a leucine (Leu38) that packs against the indole ring of the bound tryptophan in TrpE. The Tyr48 side chain is held in place by a hydrogen bond to Asp399 (Asp385 in Irp9), which also forms a double salt bridge with Arg235. In Arg235 is another key substitution, as it replaces Tyr292 in TrpE and Trp241 in PabB, which help form the tryptophan site. Arg223 in Irp9 is equivalent to Arg235 in MbtI and forms an equivalent salt bridge. In both salicylate synthases, the tryptophan binding site that is conserved among other chorismate-utilizing enzymes has clearly been ablated and replaced by a tight network of hydrogen-bonded residues, although the residues used are different in MbtI than in Irp9.

Oligomerization interface. Although structurally homologous, members of the group of chorismate-utilizing enzymes exemplified by MbtI, TrpE, and PabB show intriguing differences in oligomerization behavior. Anthranilate synthase is a heterotetramer (stoichiometry, TrpE₂:TrpG₂) with the TrpE and TrpG (amidotransferase) subunits forming a tight heterodimer, whereas PabB forms only a transient and structurally uncharacterized complex with its equivalent amidotransferase, PabA. As there is no requirement for an amidotransferase activity in the conversion of chorismate to salicylate, it is unsurprising that the TrpG-binding interface of TrpE has not been conserved in MbtI. TrpE binds to TrpG primarily via its 7 helix, with minor contributions from the 4-ß10, ß21-11, and ß4b-2 loops, the last of which is much truncated in MbtI, PabB, and Irp9. In particular, specific interactions made with TrpG by Met364, Asp367, Arg370, and Asn371 of TrpE would not be possible in MbtI. Additionally, the electrostatic potentials of the protein surfaces in this region are rather different, especially at the C-terminal end of helix 7, which is markedly more negatively charged in MbtI due to the presence of Glu307, Glu308, and Asp311.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The production of the salicylate-based siderophore mycobactin appears to be a critical component of virulence in M. tuberculosis, as its ability to scavenge iron while in the macrophage phagosome sets it apart from nonpathogenic mycobacterial species (53). The TrpE homologue MbtI has been identified by genetic organization and gene regulation to be a likely candidate for salicylate production in M. tuberculosis. Here we present structural and in vitro biochemical evidence that MbtI is a salicylate synthase, capable of both ring isomerization and pyruvate lyase activity. A bound molecule of pyruvate identified in the active site indicated that the enzyme in the form crystallized had presumably metabolized chorismate from the expression host and that pyruvate lyase activity was likely to have occurred. This activity was confirmed by using fluorimetry, NMR, and HPLC-MS to detect salicylate production in vitro. This identifies salicylate biosynthesis in M. tuberculosis as being a single-step process like that seen with Y. enterocolitica (29, 30, 41) rather than the two-step process characterized for P. aeruginosa, as had been speculated previously (18, 44). This confirmation that M. tuberculosis carries out a single-step salicylate synthesis is consistent with the original biochemical evidence for a bacterial salicylate biosynthetic pathway, which came from the identification of shikimate-derived salicylate in M. smegmatis, where the presumed two components involved in its production were unable to be separated (35).

Although several structures are now available for chorismate-utilizing enzymes, many questions remain about the mechanisms involved in the reactions they catalyze. One unanswered question is why some of the enzymes (PabB and PchA) require an additional lyase (PabC and PchB, respectively) to remove the pyruvate moiety from the reaction intermediate, while other enzymes (MbtI, TrpE, and Irp9) are able to catalyze this step without assistance. The pyruvate lyase enzymes PabC and PchB are themselves not related and are proposed to work via quite distinct mechanisms: the former is a pyridoxal phosphate-containing enzyme related to amino acid aminotransferases (24), while the latter shows weak chorismate mutase activity and is related to AroQ-type chorismate mutases (17). It has been suggested that the presence of Lys274 in PabB may both influence the specificity of the reaction and predict the need for a separate lyase (39). Arguing against this being so, PchA has an alanine residue in the equivalent position, in common with the other lyase-competent enzymes TrpE, MbtI, and Irp9. Another a priori possibility is that substitutions at position 2 of the chorismate ring, as seen in anthranilate and salicylate synthases, simply form a less chemically stable intermediate, which itself rearranges to form the reaction products. The fact that PchB is required for lyase activity in P. aeruginosa argues against this also, but it is interesting in this context to note that recent work with PchB has indicated the likelihood of the reaction proceeding via an unusual pericyclic mechanism (11, 33) rather than by the previously proposed general base proton abstraction and elimination reaction (54). Site-directed mutational analysis supports the idea that the same pericyclic mechanism is used in Irp9 (29), and by extension this is likely to be the case in MbtI also. From a structural perspective and in the absence of a structure of PchA, it is difficult to make any definitive comment on what the critical determinant of lyase capability might be, as all of the critical residues thought to be involved in the catalytic process are conserved between the lyase-competent MbtI and Irp9 and the lyase-dependent PchA.

The proposed pericyclic mechanism for lyase activity does not require a general base but does require a positively charged residue to stabilize a negative charge on the ether oxygen of the isochorismate intermediate (33). In the structure of MbtI, Lys438 is hydrogen bonded to the ketone (O3) oxygen of the bound pyruvate (Fig. 5C), which would have formed the ether bridge in the isochorismate intermediate. This residue is absolutely conserved, though it does not make any direct interactions with substrate or products in any of the other known structures. Its position, and the observation that it is able to make a hydrogen bond to the oxygen in question, makes it a plausible candidate for contributing the stabilizing positive charge required by this mechanism.

Although MbtI appears to be a monomeric enzyme in solution, in the crystal structure it makes an intersubunit contact that is equivalent to the dimerization interface of Irp9 and of S. marcescens TrpE, involving the helices 6 and 7 and beta strand ß16. As active site residues are contributed from 7 and from the adjacent ß17 strand, it is possible that enzyme activity may be influenced by multimeric assembly. Although the structure of the enzyme has been described in terms of two subdomains, each constituting a large ß-sheet, it could equally well be described as being composed of two domains, a smaller one made up from helices 6 and 7 and ß-strands ß14, ß16, and ß17 (Fig. 3) and a larger domain made up of the rest of the protein. There is inherent, functionally significant flexibility apparent in the region, as seen here with MbtI and also with PabB, where the ß14-ß16-ß17 sheet can place the active site into open and closed conformations (Fig. 5A).

Given the critical importance of iron acquisition for pathogenicity in M. tuberculosis, siderophore biosynthesis represents a clearly validated target for the development of new antituberculosis drugs. Salicyl-AMS, an inhibitor of MbtA, a bifunctional salicyl-AMP ligase/salicyl-S-ArCP synthetase which transfers salicylate onto the aroyl carrier protein (ArCP), has recently been synthesized and showed marked in vitro growth inhibition of M. tuberculosis, with 50% inhibitory concentration values in the µM range (16). The structure of MbtI presented here represents the starting point for the rational design of small molecule inhibitors of salicylate synthesis in M. tuberculosis. An initial series of chorismate-based competitive inhibitors of Irp9 has recently been described (40), the most potent of which had a K_i of 20 µM, which may provide a guide for the synthesis of specific inhibitors of MbtI.

 
We thank Didier Nurizzo (European Synchrotron Radiation Facility) for native X-ray data collection and Michael Walker (Department of Chemistry, University of Auckland) for NMR data collection.

This work was funded by the Health Research Council of New Zealand and a New Economy Research Fund grant from the New Zealand Foundation for Research, Science and Technology.

 
* Corresponding author. Mailing address: School of Biological Sciences, University of Auckland, Private Bag 92-019, Auckland 1020, New Zealand. Phone: 64-9-373-7599. Fax: 64-9-373-7414. E-mail: s.lott{at}auckland.ac.nz.

Present address: IBG, Uppsala Biomedicinska Center, Uppsala University, Box 592, S-75124 Uppsala, Sweden.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, September 2006, p. 6081-6091, Vol. 188, No. 17
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