Allosteric Regulation of Vitamin K2 Biosynthesis in a Human Pathogen

Menaquinone (Vitamin K2) plays a vital role in energy generation and environmental adaptation in many bacteria, including Mycobacterium tuberculosis (Mtb). Although menaquinone levels are known to be tightly linked to the redox/energy status of the cell, the regulatory mechanisms underpinning this phenomenon are unclear. The first committed step in menaquinone biosynthesis is catalyzed by MenD, a thiamine diphosphate-dependent enzyme comprising three domains. Domains I and III form the MenD active site, but no function has yet been ascribed to domain II. Here we show the last cytosolicmetabolite in the menaquinone biosynthesis pathway (1,4-dihydroxy-2-napthoic acid, DHNA) binds to domain II of Mtb-MenD and inhibits enzyme activity. We identified three arginine residues (Arg97, Arg277 and Arg303) that are important for both enzyme activity and the feedback inhibition by DHNA: Arg277 appears to be particularly important for signal propagation from the allosteric site to the active site. This is the first evidence of feedback regulation of the menaquinone biosynthesis pathway in bacteria, unravelling a protein level regulatory mechanism for control of menaquinone levels within the cell.


Introduction
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans, is able to adopt a persistent phenotype, resulting in long treatment times and a hard-toeradicate latent infection [1]. To combat this latent state, there has been a growing interest in menaquinone (vitamin K2, MK), a small redox molecule that is essential for energy generation in both actively growing and persistent Mtb [2]. MK also plays a role in triggering persistence in Mtb through its capacity to signal redox status [3]. Previous studies have shown that inhibiton of MK biosynthesis enzymes can significantly reduce growth of persistent-state like and drug-resistant Mtb [4][5][6]. Therefore, a fundamental understanding of the MK biosynthesis pathway and its regulatory mechanism would provide a deeper insight into the underlying complexity of Mtb biology, opening up novel approaches for anti-TB therapeutics.
MK levels are known to be tightly linked to the redox potential in bacteria [2,3,[7][8][9][10]; however, the molecular mechanisms that regulate this phenomenon are unclear. The first committed step in MK biosynthesis in Mtb is catalyzed by the thiamine-diphosphate (ThDP)-dependent enzyme MenD (2-succinyl-5-enolpyruvyl-6hydroxy-3-cyclohexadiene-1-carboxylate synthase, SEPHCHC synthase). Like other members of the ThDP-dependent pyruvate oxidase (POX) family, which are dimers or tetramers (comprising two interfacing dimers), MenD is tetrameric, with each monomer comprising three domains [11,12]. Domains I and III have known roles in catalytic function, domain I from one monomer in the dimer pairs with domain III of the other monomer (and vice versa) to form two paired active sites per dimer, with residues from both domains contributing to each active site [13][14][15]. Domain II, however, is much less conserved and does not appear to participate in cofactor or substrate binding [11,15].
We previously determined a series of crystal structures of Mtb-MenD showing each step in the MenD catalytic cycle, as substrates a-ketoglutarate and isochorismate are successively added to the ThDP cofactor before the final product is released [13].
We have now identified a downstream metabolite of the MK biosynthesis pathway (1,4-dihydroxy-2-napthoic acid, DHNA) that binds to domain II of Mtb-MenD and inhibits its catalytic activity. Herein we characterize DHNA binding to Mtb-MenD at the molecular level, providing evidence for protein-level allosteric regulation and feedback inhibition of the classical MK biosynthesis pathway in Mtb and opening up new and unanticipated possibilities for therapeutic intervention in this important pathway.

Mtb-MenD
The classical MK biosynthesis pathway ( Figure 1A) starts with the synthesis of the napthoquinone head-group precursor (DHNA) in the cytosol [22,23], followed by prenylation and methylation by membrane bound enzymes to produce the lipid soluble MK [24,25]. In addition to its electron transport role, the prenyl tail of MK can be further modified and these modified quinones have been shown to regulate virulence of the Mtb infection [7,26,27]. Mtb-MenD is situated at a key step in MK biosynthesis, the first committed step (Figure 1A/B) [28,29] and we hypothesized that it might be involved in controlling flux though this pathway. In particular, we aimed to determine whether Mtb-MenD is subject to feedback regulation. We soaked crystals of Mtb-MenD with a range of substrates, metabolites, and metabolite-like compounds from the MK biosynthesis pathway to determine whether any of them bound to the enzyme, and found that DHNA, a downstream metabolite of MK biosynthesis, binds to a site in domain II of Mtb-MenD ( Figure 1C).  (Figure 2A). This cleft is located ~ 20 Å away from the closer of the paired active sites in the dimer and ~ 30 Å from the more distant one (Figure 1C and 2B).

To further characterize the interactions between
The binding site for DHNA (Figure 2A)  Whether there is any connectivity between the four allosteric DHNA binding sites in the Mtb-MenD tetramer (as there is between the active sites [13]) is unclear.
However, the domain II residues 299-306 at the DHNA binding site are connected by a hydrogen bonding network involving residues Arg97, Ala170, Arg159 and Arg168 to the same region in a neighboring Mtb-MenD subunit ~25 Å away. This suggests that binding events on one subunit could be transmitted to the others through such a network.
The location of the DHNA binding site is similar to that of allosteric activator pockets present in another member of the POX superfamily, pyruvate decarboxylase [30]; therefore, we carried out assays to determine whether DHNA acts in a similar manner, as an allosteric regulator of MK biosynthesis.

DHNA is a potent inhibitor of Mtb-MenD SEPHCHC synthase activity
To investigate whether DHNA plays a regulatory role in MK biosynthesis, we studied its effect on Mtb-MenD activity. An NMR-based activity assay, similar to that previously reported [13], was first used and showed that the activity of Mtb-MenD at a concentration of 5 µM was reduced in the presence of 20 µM DHNA to only 24% of its non-inhibited activity ( Figure 3A). Further increases in DHNA concentration resulted in only small increases in inhibition (data not shown), consistent with saturation of the enzyme at low micromolar concentrations.
A UV spectrophotometry-based assay, in which the consumption of isochorismate is monitored at 278 nm, was then used to examine the inhibition over a lower DHNA concentration range (0.1 nM to 10 µM). We found that DHNA inhibited Mtb-MenD with an IC50 of 53 nM under the conditions of this assay ( Figure 3B, Table   1). In combination with our structural complexes, these assays establish DHNA as a potent allosteric inhibitor of Mtb-MenD.

Connectivity between the allosteric and active sites of Mtb-MenD
How might binding at a site that is remote from the active site impact on catalysis? Mtb-MenD is a complex enzyme, characterized by significant conformational changes and disorder-order transitions that take place during the catalytic cycle [13]. In its apo (cofactor-free) state there are substantial regions of disorder. Cofactor-bound structures, including the two covalent intermediates, are more ordered and are also asymmetric, with only two of the four active sites occupied per tetramer [13]. The most striking conformational change associated with DHNA binding involves a flexible active site loop, residues 105-125, that carries the substrate binding residues Arg107, Asn117 and Gln118. This loop is disordered in the apo state, but becomes fully ordered when its associated active site is occupied ( Figure 4A). Also reorganized are residues 79-82 at the N-terminus of an a-helix that helps form the binding pocket for the 4'aminopyrimidine (AP) ring of the ThDP cofactor. These changes are required to generate the catalytically-competent state of the enzyme, and presumably also to enable product release.
The DHNA binding site is ~20 Å from the closest active site, with the two sites separated primarily by two sections of polypeptide, residues 399-402 from domain III and residues 114-120 from the flexible active site loop contributed by the other subunit of the dimer ( Figure 2B). Residues 114-120 carry two key active site residues, Gln118, essential for catalysis [13], and Asn117, which binds to the a-ketoglutarate moiety when intermediate I is formed. Similarly, the 399-402 loop provides Arg399, critical for a-ketoglutarate recognition, and Ala402, whose carbonyl oxygen hydrogen bonds to the ThDP AP ring ( Figure 2B). DHNA binding to the apo-enzyme causes residues 114-120 to become fully ordered as they complete its binding site, and is associated also with reorganization of residues 78-82, as a new hydrogen bond is made between Ser79 and Gln118 ( Figure 4B). The hydrogen bonding environment of the catalytically-essential Glu55 is also changed as a result, to a state part-way between that seen in occupied and unoccupied active sites. This occurs for two of the four active sites creating asymmetry previously only observed in co-factor occupied structures. In many respects the structural changes that occur as DHNA binds to the apo-enzyme thus mirror key changes in domain I that occur when ThDP binds.
From the above it is clear that there is connectivity between the two sites such that the allosteric site is affected by, and can influence, the active site of Mtb-MenD. A key player in this scenario is likely the invariant Gln118, even conservative mutations of which abolish SEPHCHC synthase activity [31]. In unoccupied active sites, Gln118 is disordered or has high B factors. In occupied sites, however, it is ordered but undergoes sidechain movements that enable specific interactions that are critical to several key steps of the catalytic cycle. These include stabilizing the active tautomer of the AP ring and hydrogen bonding to both the incoming isochorismate substrate and then the resultant intermediate II. It also interacts with the "CO2-like" formate ion that likely models the location of the carboxyl group that is removed during formation of intermediate I [13,32].
The three arginine cage residues support inhibition by DHNA and dramatically affect enzyme activity The three arginine residues (Arg97, Arg277, and Arg303) that form a cage around DHNA in its binding site are candidates for signaling between the allosteric and active sites. All three residues interact directly with DHNA and are likely to enhance binding (Figure 2A/B). Arg97 is located at the C-terminus of a long helix that originates in the more distant of the paired active sites, indicating a potential line of communication with that site (Figure 2A/B). Arg277 hydrogen bonds with two regions of the closer of the paired active sites, i.e. with Gly400/Arg399, and with two residues from the 105-125 active site loop ( Figure 2B). These regions contain residues (Arg107, Asn117, Gln118, and Arg399) known to be important for Mtb-MenD function [28,32]. Arg303 is located in a region (residues 299-308) that interacts with another part of the 105-125 loop ( Figure 2A) and is also involved in a hydrogen bonding network with the closest allosteric site across the tetramer.
To test the importance of these three residues for either MenD activity and/or DHNA inhibition, we carried out alanine mutagenesis experiments. These confirmed that each of the three arginines is crucial for MenD activity ( Table 1): the R303A, R97A, and R277A mutant proteins had 56%, 50%, and 18%, of the wild type (WT) enzyme activity, respectively, when measured under the same conditions and in the absence of DHNA. In terms of DHNA inhibition, R97A and R303A showed 19-fold and 6-fold increases in IC50, respectively, compared to WT Mtb-MenD (Table 1) indicating the importance of these arginines for DHNA binding and feedback inhibition. The R277A variant had such a low catalytic activity that its IC50 could not be measured. We conclude that these three residues are important for maintenance of WT MenD activity, probably because they stabilize other elements of the active site (e.g. the 105-125 loop) and that this underpins their roles in signal propagation from the allosteric site to the active site of Mtb-MenD.

How conserved is this allosteric site in other MenD enzymes?
To explore how widely conserved the newly discovered allosteric site is across the bacterial kingdom, we analysed both sequence and structural conservation of this site within MenD orthologues (Figure 5B/5C Our studies suggest there is limited strict conservation of key elements of the allosteric site across bacterial MenD enzymes, which could indicate the regulation of the pathway by DHNA is limited to a small sub-set of bacteria. However, considering the partial conservation in some organisms, it is possible that DHNA or related molecules could still bind in this region despite the absence of some key residues found in Mtb-MenD.
Exactly how widespread allosteric regulation of menaquinone biosynthesis is among bacteria, therefore, remains a question for future work. However the presence of a site with a powerful ability to regulate enzyme activity is of immediate value as a target for species-specific antimicrobials.

Domain II is adapted for regulatory roles in other ThDP-dependent enzymes
Our results demonstrate that Mtb-MenD is an allosterically-regulated ThDP-dependent enzyme, inhibited by direct binding of a downstream metabolite (DHNA) from the biosynthetic pathway in which it participates. While feedback inhibition of this type has not been demonstrated for ThDP-dependent enzymes before, there is precedence for various mechanisms of allosteric regulation in the wider superfamily. Some enzymes, such as acetohydroxyacid synthase, have an entirely distinct negative regulatory subunit [34]. Others, such as 2-ketoglutarate dehydrogenase (KGD) and 2hydroxy-3-oxoadipate synthase, are allosterically activated by binding of a small molecule (acetyl-CoA) to a shallow pocket on the enzyme surface and allosterically inhibited by binding of a regulatory protein (GarA) [35][36][37]. Similarly, pyruvate decarboxylase [30], phenylpyruvate decarboxylase [17,18], and oxalyl-CoA decarboxylase [21] have all been shown to be positively regulated by the direct binding of small molecules to domain II.
Structural comparisons of DHNA-bound Mtb-MenD reveal striking parallels with pyruvate decarboxylase, which is allosterically activated by its pyruvate substrate [30]. The pyruvate decarboxylase allosteric site has previously been described as a "switch point" in domain II [30] and has a very similar structural context to the DHNA- Despite these similarities, key differences exist. The allosteric effect in pyruvate decarboxylase is activation not inhibition, and, in keeping with this, the effector of Mtb-MenD is not the substrate but a downstream metabolite. In addition, while the asymmetric dimers of pyruvate decarboxylase undergo large quaternary conformational changes [38], no significant alterations in quaternary structure have been observed for any MenD structures reported to date. The enzymes also diverge in function, catalyzing reactions with different sized substrates and requiring different dynamics during their catalytic cycles [18,30]. Nonetheless, these parallels do point to the wider use of domain II as a regulatory domain for allosteric regulation of ThDPdependent enzymes, a phenomenon now also seen in MenD.

The biological significance of a regulatory role for DHNA
The ability of DHNA to act as a regulatory signal in the pathogen M. tuberculosis is in line with both the biological significance of DHNA and the importance of regulating menaquinone levels within the bacteria. As the last non-prenylated soluble metabolite in the MK biosynthetic pathway, DHNA sits at the point where the pathway moves from an aqueous cytosolic location to a lipophilic membrane-immersed one [25] and has the potential to provide feedback on the catalytic status of MenA (and perhaps the downstream MK pool). DHNA is also the first metabolite in the pathway with a complete redox-capable napthoquinone ring [39], and has the capacity in its own right to catalyze redox reactions [40]. It may thus act as a signal of redox status, with excessive levels exerting toxicity if the redox balance within the cell is disrupted.
DHNA has also been shown to act as a virulence factor in the intracellular pathogen Listeria monocytogenes, where it promotes cytosolic survival, and may be a sensor of cytosolic stress [41]. In plants, phylloquinone biosynthesis enzymes share homology to those of classical bacterial MK biosynthetic enzymes and 1,4 naphthoquinones derived from DHNA act in roles mediating plant-plant, plant-insect and plant-microbe interactions [42].

Conclusions
Our study reveals the downstream metabolite DHNA as a negative allosteric regulator

Strains and plasmids
The open reading frame encoding MenD (Rv0555) from M. tuberculosis H37Rv was previously cloned into the pYUB28b vector [13]. MenD mutants were generated using the pYUB28b-menD construct and oligonucleotide primers (Supplementary Table 2, Integrated DNA Technologies) with iProof TM high-fidelity DNA polymerase (Bio-Rad). The PCR products were then treated with DpnI and ligated using T4 DNA ligase (Roche), before being transformed into E. coli TOP10 cells. The mutations were verified by DNA sequencing.

Protein expression and purification
WT and mutant MenD constructs were expressed in M. smegmatis mc 2 4517 cells and purified using immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC) as described previously [13]. In brief, cells were lysed in For NMR experiments, the protein was either purified directly into 50 mM phosphate pH 7.5 with 50-100 mM NaCl or buffer-exchanged into this after purification.

NMR spectroscopy assay
The activity of Mtb-MenD was monitored using a coupled reaction with E. coli isochorismate synthase (Ec-MenF), which converts chorismate to isochorismate (the substrate for Mtb-MenD). Ec-MenF was expressed and purified as previously described [13]. Initial NMR samples were prepared with 2 mM chorismate, 1 mM a- were incubated at 25 ºC and 1D 1 H NMR spectra monitored until the reaction reached equilibrium, with an estimated 47:53 ratio of isochorismate to chorismate based on peak integrals. Mtb-MenD (5 µM) was then added and 1D 1 H NMR spectra were recorded at 100 s intervals for up to 90 min. Reaction rates were estimated by monitoring the decrease in peak integral for isochorismate and chorismate, and the increase in peak integrals for SEPHCHC, relative to the peak for the TSP internal standard (d 0 ppm).
Due to peak overlap in other regions, the pyruvyl methylene proton peak was monitored with a chemical shift of 5.17, 5.15 and 5.12 ppm for isochorismate, chorismate and SEPHCHC, respectively. NMR spectra were collected on an Avance AVIII-HD 500 MHz Spectrometer (Bruker) with suppression of the water signal using excitation sculpting [43]. Data were processed using the software package TopSpin 4.0.6 (Bruker).

UV-Visible spectroscopy assay
Mtb-MenD activity was monitored by the decrease in isochorismate absorbance at 278 nm (ε278 = 8,300 M -1 .cm -1 ; [29]) at 25 °C. To produce isochorismate, 10 µM Ec-MenF was incubated in a 3 mL reaction for 2 h at room temperature with 100 mM Tris HCl pH 7.5, 100 mM NaCl, 5 mM MgCl2 and at least 3 mg chorismic acid. Ec-MenF was then removed from the mixture using a vivaspin concentrator with a 10 kDa cutoff. The mixture was stored in small aliquots at -80°C prior to use.
Isochorismate was quantified prior to kinetic assays, using the following reaction: 1 µM Mtb-MenD was incubated with 100 µM thiamine pyrophosphate (ThDP) and 100 µM a-ketoglutarate for 30 min at 25 °C in MenD kinetic assay buffer (100 mM Tris pH 8, 100 mM NaCl and 5 mM MgCl2). The reaction was initiated with 30 µl of a mixture of chorismate/isochorismate. The quantity of isochorismate used was back-calculated using Beer's law. No background catalytic rate was observed when either isochorismate or a-ketoglutarate were absent from the assay mixture. All assays were performed using a Cary 400 UV-VIS spectrophotometer and quartz cuvettes with a final reaction volume of 800 µl.

Data collection, structure determination and refinement
All diffraction data were collected using the macromolecular crystallography beamline MX2 at the Australian Synchrotron. The data were indexed and processed using XDS [45], re-indexed using POINTLESS [46] and scaled with SCALA [46] from the CCP4 program suite [47]. Analyses of merged CC½ correlations between intensity estimates from half data sets were used to influence high resolution cutoff for data processing [48].
The structures of the DHNA-soaked crystals were solved by molecular replacement using Phaser [49], with 5ERY [13] as a search model, and a dimer of a previous Mtb-MenD structure (PDB code: 5ESU [13]) was used as a search model for the ThDP/Intermediate structures. The final models of all structures were then completed with iterative rounds of manual building using COOT [50] and refinement using Refmac5 [47] and Phenix [51]. After building, additional density corresponding to DHNA, ThDP, intermediate I or II, and a-ketoglutarate, as appropriate, was modelled using available PDB dictionary restraints. Water molecules were identified by their spherical electron density and appropriate hydrogen bond geometry with the surrounding structure. Unless otherwise stated all protein structure images were generated using Pymol (The PyMOL Molecular Graphics System, Version 1.5, Schrödinger, LLC).   In the presence of DHNA the rate of production of SEPHCHC (d 5.20 ppm) was 24 ± 1% of that in its absence. The doublet peaks assigned to isochorismate, chorismate and SEPHCHC correspond to the equivalent methylene hydrogen (H8b) on the enolpyruvyl group of each compound. Full 1 H NMR assignment of SEPHCHC was reported previously [13]. B. IC50 for DHNA against WT Mtb-MenD measured using a UV spectrophotometry based-assay for isochorismate consumption. Initial inhibition assay solutions contained 0.6 µM Mtb-MenD, 300 µM ThDP and various concentrations of DHNA (0 to 10 µM) and were pre-incubated for 30 min at 25°C. Then 2 µM isochorismate was added and the reaction was initiated by the addition of 300 µM of a-ketoglutarate. Initial rates were measured and fit to the four-parameter logistic Hill equation (solid line).