An essential bifunctional enzyme in Mycobacterium tuberculosis for itaconate dissimilation and leucine catabolism

Significance Accounting for 1.3 million deaths in 2017, the bacillus Mycobacterium tuberculosis (Mtb) primarily resides within human macrophages. Itaconate is suggested to be an antimicrobial metabolite and immunomodulator produced by macrophages during Mtb infection. Here, we show that Mtb is intrinsically resistant to itaconate and Mtb degrades l-leucine via an unprecedented R-stereospecific route. Our work reveals Rv2498c as a bifunctional enzyme involved in itaconate dissimilation and l-leucine catabolism in Mtb.

was cloned by isothermal assembly (HiFi DNA Assembly, NEB) into a modified pET28a vector (Merck) coding for an N-terminal His6 tag followed by a TEV protease cleavage site. The construct was verified by Sanger sequencing.
E. coli BL21 Star (DE3) (ThermoFisher) cells were transformed with the construct. A single colony was used to inoculate 10 mL of lysogeny broth. The cell pellet obtained after overnight incubation (37 °C and 200 rpm) was diluted 50X into autoinduction medium (ZYM-5052/60 μg/mL kanamycin). Cells were grown for 4 hours at 37 °C followed by 20 hours at 18 °C.

Phylogenetic analysis
A seed multiple sequence alignment (MSA) of selected members of the HpcH_HpaI aldolase/citrate lyase family (PF03328) was built based on an alignment of the structures available in the PDB. (2) Structures were visualised and edited using Chimera and the multiple structural alignments was computed by Mustang. (3,4) The PF03328 sequences in the fullalignment were downloaded from PFAM and aligned to the seed MSA using MAFFT E-INS-I-add option.(5) Partial and highly divergent sequences were removed and redundancy was reduced to 90% ID or less using Jalview. (6) The model of protein evolution used was LG+I+G+F with α of 1.64 and p-inv of 0.01, as selected by Prottest 3.4.(7) Bootstrap repeats and consensus trees were generated with PHYLIP. (8) Pairwise distances were calculated with TREEPUZZLE via the puzzleboot script. (9) Distance trees were calculated with BIONJ. (10) For the acyl-CoA lyase tree, sequences were aligned with MUSCLE and a maximumlikelihood tree was calculated with PhyML. (11,12) Trees were drawn with Dendroscope 2.7. (13)

Mtb spotting culture
The spotting culture is essentially the conventional bacteria growth on agar medium without streaking the bacteria. Mtb liquid cultures were grown in 7H9 medium supplemented with Tyloxapol and ADC to OD600 of 1. Serial dilutions of 1:5. 2 µL of the OD600 of 1 and the diluted cultures were spotted on chemically defined-7H10 medium with test compounds (e.g. L-leucine, itaconate, L-valine, L-isoleucine, acetate, oleate, pyruvate… etc.). The plates were incubated at 37 °C. Growth phenotypes were observed, and images were taken on day 25 post spotting.

Cell-free protein extract preparation
Mtb liquid culture was grown at 37 °C in 7H9 medium supplemented with Tyloxapol and ADC to OD600 of 1. The cell pellet was collected after centrifugation at 2,000 rpm for 10 min at 4 °C, and then washed with HEPES (50 mM, pH 7.5, 5 mM MgCl2). The washed cell pellet was resuspended in the HEPES buffer containing cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche) and transferred to O-ring screw cap tube with glass beads (150-212 µm, acid washed, Sigma), and ribolysed at 2 x 30 sec with cooling in between the two cycles. The clear supernatant was collected after centrifugation at 13,000 rpm for 10 min at 4 °C, and then filtered twice through 0.2 µm membrane. The total protein concentration was determined by BCA assay (Pierce).

Murine aerosol M. tuberculosis infections
C57BL/6J (WT) mice were bred and maintained under specific pathogen-free conditions at The Francis Crick Institute, Mill Hill Lab. Animal studies and breeding were approved by the Francis Crick Institute ethical committee and performed under U.K Home Office project license PPL 70/8045. Infections were performed in the category 3 animal facility. For mouse infections, M. tuberculosis strains were cultured in Middlebrook 7H9 broth containing ADC to an OD600 of 0.6. From this, an infection sample was prepared to enable delivery of 100 colony forming units (CFUs)/mouse lung using a modified Glass-Col aerosol infection system. Infection was monitored by assessing homogenised lungs from infected mice at defined periods post-infection. Bacterial CFUs were determined by plating serial dilutions of homogenates on duplicate Middlebrook 7H11 containing OADC. Colonies were counted 2-3 weeks after incubation at 37 º C. The data at each time point are the means of 5 mice/group +/-SEM.

Filter culture and metabolite preparation
The filter culture followed the protocol as described by Carvalho et al., with minor modifications. (16) Mtb was cultivated in 7H9 medium supplemented with Tyloxapol and ADC at 37 °C to reach an OD600 of 1. A total of 1-mL of the liquid culture was transferred to a membrane disc under vacuum to collect the cells on the membrane. The membrane disc containing the cells was transferred on 7H10 medium supplemented with OADC and incubated for 5-6 days until sufficient cell mass has accumulated. The membrane disc containing the cell mass was then transferred to a conditioned-7H10 minimum medium supplemented with relevant test substrate and incubated for an additional 17 hr maximum to avoid doubling of the cells. After the 17 hr exposure, the cells were scrapped and transferred to an O-ring screw cap tube with glass beads (150-212 µm, acid washed, Sigma), with the addition of 0.5-0.7 mL cold acetonitrile:methanol:water (2:2:1) and ribolysed for 2 x 30 sec with cooling between the two cycles. The clear supernatant was collected after centrifugation at 13,000 rpm for 10 min at 4 °C and filtered twice through 0.2 µm membrane. The filtered supernatant was analysed by LC/MS or stored at -80 °C until further use.

LC-MS metabolomics
The clear supernatants from metabolite preparation were directly analysed by the LC-MS method described by Larrouy-Maumus et al. (17) Briefly, an Agilent 1200 LC system consisting of a solvent degasser, binary pump, temperature-controlled auto-sampler and temperature-controlled column compartment equipped with a Cogent Diamond Hydride Type C silica column (150 mm × 2.1 mm; dead volume 315 μL) was used for liquid chromatography. The flow rate was 0.4 ml/min. Solvent A was 0.2% acetic acid in water and solvent B was 0.2% acetic acid in acetonitrile. The gradient was as follows: 0-2 min 85%B; 2-3 min to 80%B; 3-5 min 80%B; 5-6 min to 75%B; 6-7 min 75%B; 7-8 min to 70%B; 8-9 min 70%B; 9-10 min to 50%B; 10-11 min 50%B; 11-11.1 min to 20%B; 11.1-14 min hold 20%B. (18) An Agilent Accurate Mass 6230 TOF apparatus was used. Dynamic mass axis calibration was achieved by continuous infusion of a reference mass solution using an isocratic pump connected to a multimode ionization source, operated in the positive-ion and negative-ion mode. ESI capillary and fragmentor voltages were set at 3500 V and 100 V, respectively. The nebulizer pressure was set at 40 psi and the nitrogen drying gas flow rate was set at 10 L/min. The drying gas temperature was maintained at 250°C. The MS acquisition rate was 1.5 spectra/sec and m/z data ranging from 50-1200 were stored. The instrument routinely enabled accurate mass spectral measurements with an error of less than 5 parts-per-million (ppm), mass resolution ranging from 10,000-25,000 over the m/z range of 121-955 atomic mass units, and a 100,000-fold dynamic range with picomolar sensitivity. Data were collected in the centroid mode in the 4 GHz (extended dynamic range) mode.

LC-UV/MS
LC-UV/MS analysis was performed on an Agilent 1290 LC system equipped with a diode array UV detector coupled to an Agilent 6560 Ion Mobility Q-ToF (operated in Q-ToF only mode).
Chromatography was performed using an Agilent Poroshell 120 EC-C18, 2.7 µm, 4.6 x 50 mm column (ambient temperature) and 40 mM ammonium formate, pH 6.8 and acetonitrile as mobile phases A and B respectively. Analytes were eluted using a flow rate of 0.5 mL/min and the following mobile phase gradient: 0 -1 min, 2% B; 1 -10 min, 2 -20% B; 10 -12 min, 20% B. The system was re-equilibrated to initial conditions for two minutes at the end of each run. The injection volume was 3 µL for all standards and samples. UV detection was carried out at 260 nm.
The Q-ToF was operated in negative-ion mode with electrospray ionisation (ESI) using a dual AJS ESI source. Capillary, nozzle and fragmentor voltages were set at 3000 V, 2000 V and 380 V respectively. The nebulizer pressure was set at 40 psi and the nitrogen drying gas flow rate was set at 10 L/min. The drying gas temperature was maintained at 200 °C. The sheath gas temperature and flow rate were 350 °C and 11 L/min. The MS acquisition rate was 1 spectra/sec and m/z data ranging from 50-1700 were stored. Dynamic mass axis calibration was achieved by continuous infusion of a reference mass solution, which enabled accurate mass spectral measurements with an error of less than 5 parts-per-million (ppm).

Enzyme assays
Rv2498c or cell-free protein extract (CFPE) reaction was carried out in buffer containing 50 mM HEPES (pH 7.5), 5 mM MgCl2, and specified concentration of substrate (usually in the 10-50 µM range). Reaction was initiated by the addition of enzyme or CFPE, and incubated at 37 °C for 30 minutes, 2 hours, or overnight. The reactions were terminated on ice and quenched with 1 µL of 1M HCl per 10 µL reaction volume or without quenching, and directly used for HPLC analysis. Typically, the reaction mixture was quenched and centrifuged at 13,000 rpm for 10 min at 4 °C, and the clear supernatants were used for HPLC analysis.

Measurement of enzyme kinetics
The kinetic studies were carried out in buffer containing 50 mM HEPES (pH 7.5), 5 mM MgCl2, a fixed concentration of the enzyme, and specified concentration of substrate (usually 25, 50, 100, 250, 500 µM). The reaction was initiated by the addition of the purified enzyme, with aliquots of the reaction collected at various times and quenched with HCl on ice for each concentration of the substrate to determine the forward initial velocities. The clear supernatant of the quenched samples was collected after centrifugation (13,000 rpm, 10 min, 4 C), and then injected into the HPLC. Substrate and product were measured at 260 nm, and the concentration of the reaction product was determined using an Ac-CoA standard curve. The results were fitted on Michaelis-Menten curve or Lineweaver-Burk plot to determine the Vmax and Km. The calculation for HMG-CoA lyase kinetic values took into consideration of the commercial equal molar racemic mixture of (R/S)-HMG-CoA by dividing the concentration of HMG-CoA substrate by two.
Each CoA-thioester was detected as a single peak after being separated by HPLC with a Poroshell 120 EC-C18, 2.7 µm, 4.6 x 50 mm column (Agilent) using the following elution condition: 1-min isocratic elution at 2% acetonitrile in buffer, followed by a 10-min linear gradient of 2-20% acetonitrile, with 2-min isocratic elution at 20% acetonitrile in buffer, and then back to 1-min isocratic elution at 2% acetonitrile in buffer at a flow rate of 0.5 ml/min. The buffer used was 40 mM ammonium formate, pH 6.8. For more hydrophobic CoAthioesters, 95% instead of 20% acetonitrile was used for the gradient. When necessary, CoAthioesters were quantified by a calibration curve generated from the authentic Acetyl-CoA standard (Sigma-Aldrich) stock solutions (5,15,25,50, 100, 150, 250 µM).

Pyruvate derivatization with phenylhydrazine HCl.
Pyruvate derivatization followed the method described by Lange and Mályusz with minor modifications.(19) Briefly, 1.5 molar equivalent of phenylhydrazine was added to the acid quenched CoA crude reaction mixture and incubated for 1 hr at 37 °C. The clear supernatant of the reaction mixture was collected after centrifugation and then injected in the HPLC for analysis. The pyruvate-phenylhydrazone is monitored as 324 nm.

Construction of Mtb rv2498cKO mutant and complements
The construction of Rv2498c knockout followed methods described by Parish et al. (20) Briefly, an unmarked in-frame deletion of the Rv2498c gene was made by amplifying 1.5 kb of flanking sequence from Mtb H37Rv with Phusion Taq (upstream of Rv2498c: 5'-GCAGATCTTCGGCGGCCATCGCGTCGTA-3', 5'-GCTCTAGAACCTCCGAATGAGGGCGCAG-3'; downstream of Rv2498c: 3' for: 5'-GCTCTAGAACGCAGGTTCATTGCGCCTC-3', 5'-GCAGATCTTGGTACTTGAGGAGCTGGGC-3'), and cloned into PCR4blunt (Invitrogen). The inserts digested with BglII and XbaI, and the fragments were ligated together using T4 DNA ligase. The ligation product was amplified (5'-GCAGATCTTCGGCGGCCATCGCGTCGTA-3', 5'-GCTCTAGAACGCAGGTTCATTGCGCCTC-3'), digested with BglII and cloned into the BamH1 site of p2NIL. The PacI fragment of pGOAL17 containing the lacZ and sacB genes was cloned into the PacI site of the resulting plasmid to make the final rv2498cKO construct plasmid. Competent Mtb H37Rv was electroporated with the knockout construct plasmid, and single crossovers were selected on 7H11 plates containing kanamycin and X-gal. The blue colonies were then streaked on 7H11 plates and then double crossovers were selected on 7H11 plates containing sucrose and X-gal, the resulting white colonies were screened for double crossovers. Southern blot was performed to confirm a knockout of Rv2498c.
Other techniques DNA sequencing was performed by GATC Biotech (Konstanz, Germany). Protein concentration was determined by bicinchoninic acid (BCA) assay (Pierce BCA Protein Assay Kit), using bovine serum albumin as the standard.

Crystallization Conditions
Preparations of Rv2498c containing various ligands crystallized under two general conditions, acetate pH 7 and sulphate (or phosphate) pH ~5, yielding different crystal forms (R32 or C2, respectively). Crystals were grown using the sitting drop vapour diffusion method at room temperature. The initial protein solution contained Rv2498c at a concentration of 15 mg/mL in 50 mM HEPES pH 7.4 and 300 mM NaCl.
Prior to data collection, all crystals were transferred to cryoprotectant solutions composed of their mother liquors supplemented with 20% glycerol and flash-cooled in a nitrogen stream at 100K. X-ray diffraction data were collected by LRL-CAT staff at APS beamline 31-ID-D (Advanced Photon Source, Argonne National Laboratory, Argonne, IL) on a Rayonix 225-HE detector (Rayonix). Diffraction intensities were integrated, scaled and merged with the programs DENZO and SCALEPACK. (

Crystallographic Structure Determination and Refinement
Non-CoA ligands: The structures of three of the complexes, Rv2498c•Mg 2+ •Acetate, Rv2498c•Mg 2+ •Acetoacetate, and Rv2498c•Mg 2+ •Pyruvate, were solved by molecular replacement using PHENIX with the apo structure of Rv2498c from M. tuberculosis (PDB ID 1U5H; residues 1-221) as a search model. (22,23) Automatic model building with ARP/wARP was followed by iterative cycles of manual model building and refinement, performed with COOT and PHENIX, respectively. ( Table S2.
The final models of 6CHU, 6CJ3 and 6CJ4 consist of the following: 6CHU -amino acids 1-221 and 250-265 of Rv2498c and one His of the N-terminal affinity tag. All other cloning artifacts along with amino acids 222-249 and 266 to 273 were not observed in the electron density, presumably due to disorder. One magnesium ion and four acetate molecules were also well ordered in the structure and were included in the final model.
6CJ4 -amino acids 1-224 and 251-265 of Rv2498c. All cloning artifacts along with amino acids 225-250 and 266 to 273 were not observed in the electron density, presumably due to disorder. One magnesium ion, one acetate, and an acetoacetate molecule were also well ordered in the structure and were included in the final model. 6CJ3 -amino acids 2-224 and 250-265 of Rv2498c. All cloning artifacts along with amino acids 1, 225-249 and 266 to 273 were not observed in the electron density, presumably due to disorder. One magnesium ion, three acetates, and two pyruvate molecules were also well ordered in the structure and were included in the final model.

Rv2498c·Mg 2+ ·CoA·Acetoacetate:
The complex structure of Rv2498c with Mg 2+ , acetoacetate and CoA was determined by molecular replacement with PHENIX using the complex of Rv2498c with Mg 2+ and acetoacetate, determined earlier, as a search model. (22,23) Several rounds of automated and manual building and refinement, with COOT, ARP/wARP, and PHENIX, respectively, converged with Rwork=0.217 and Rfree=0.277 at 2.04Å resolution. (23)(24)(25) The final model consists of Rv2498c residues 3-269 in all three chains of the trimeric asymmetric unit (the N-terminal cloning artefacts, affinity tag, and residues 1, 2, and 270-273 were not observed). A magnesium ion, coordinated acetoacetate, and CoA molecule were well ordered in the active sites of three of the Rv2498c subunits and were included in the refined model. Additionally, two glycerol molecules and three sulphate ions were located and included in the final model. The coordinates and structure factors have been deposited in the PDB as entry 6AS5.
Rv2498c·Mg 2+ ·Citramalyl-CoA·Pyruvate: The complex structure of Rv2498c with Mg 2+ , pyruvate, and citramalyl-CoA was determined by molecular replacement with PHENIX using the complex of Rv2498c with Mg 2+ and pyruvate, determined earlier, as a search model. (22,23) Several rounds of manual building and refinement, with COOT and PHENIX, respectively, converged with Rwork=0.191 and Rfree=0.233 at 1.83Å resolution. (23)(24)(25) The final model consists of Rv2498c residues in three chains of the trimeric asymmetric unit (chain A, 2-268; chain B, 1-268; chain C, 1-267; the N-terminal cloning artefacts, affinity tag, and residues 1 in chain A, 269-273 in chains A and B, and 268-273 in chain C were not observed). A magnesium ion was located in each active site; two citramalyl-CoA molecules (presumably arising as the product of enzyme-mediated reaction between pyruvate and acetyl-CoA both of which were present in the crystallization milieu) were well ordered in the active sites of chains A and B and were included in the model. The magnesium ion in the active site of chain C was coordinated by a pyruvate molecule. Additionally, two glycerol molecules and three phosphate ions and five chlorides were located and included in the final model. The coordinates and structure factors have been deposited in the PDB as entry PDB entry 6AQ4.
Rv2498c·Mg 2+ ·CoA·Pyruvate: The complex structure of Rv2498c with Mg 2+ , pyruvate and CoA was solved by molecular replacement with PHENIX using the complex of Rv2498c with Mg 2+ and pyruvate, determined earlier, as a search model. (22,23) Several rounds of building and refinement, with COOT and PHENIX, respectively, converged with Rwork=0.219 and Rfree=0.262 at 1.72Å resolution. (23)(24)(25) The final model consists of Rv2498c residues 2-269 in each of three chains of the trimeric asymmetric unit (the N-terminal cloning artifacts, affinity tag, and residues 1 and 270-273 were not observed). A magnesium ion, coordinated pyruvate, and CoA molecule were observed in the active sites of three of the Rv2498c subunits and were included in the refined model. Additionally, five glycerol molecules and three phosphate ions were located and included in the final model. The coordinates and structure factors have been deposited in the PDB as entry 6ARB.
Rv2498c·Mg 2+ ·CoA·Acetoacetate: The complex structure of Rv2498c with Mg 2+ , acetoacetate and CoA was solved by molecular replacement with PHENIX using the complex of Rv2498c with Mg 2+ and acetoacetate, determined earlier, as a search model. (22,23) Several rounds of automated and manual building and refinement, with COOT, ARP/wARP, and PHENIX, respectively, converged with Rwork=0.217 and Rfree=0.277 at 2.04Å resolution. (23)(24)(25) The final model consists of Rv2498c residues 3-269 in all three chains of the trimeric asymmetric unit (the N-terminal cloning artifacts, affinity tag, and residues 1, 2, and 270-273 were not observed). A magnesium ion, coordinated acetoacetate, and CoA molecule were well ordered in the active sites of three of the Rv2498c subunits and were included in the refined model. Additionally, two glycerol molecules and three sulfate ions were located and included in the final model. The coordinates and structure factors have been deposited in the PDB as entry 6AS5.
Rv2498c·Mg 2+ ·Citramalyl-CoA·Pyruvate: The complex structure of Rv2498c with Mg 2+ , pyruvate, and citramalyl-CoA was solved by molecular replacement with PHENIX using the complex of Rv2498c with Mg 2+ and pyruvate, determined earlier, as a search model. (22,23) Several rounds of manual building and refinement, with COOT and PHENIX, respectively, converged with Rwork=0.191 and Rfree=0.233 at 1.83Å resolution. (23)(24)(25) The final model consists of Rv2498c residues in three chains of the trimeric asymmetric unit (chain A, 2-268; chain B, 1-268; chain C, 1-267; the N-terminal cloning artifacts, affinity tag, and residues 1 in chain A, 269-273 in chains A and B, and 268-273 in chain C were not observed). A magnesium ion was located in each active site; two citramalyl-CoA molecules (presumably arising as the product of enzyme-mediated reaction between pyruvate and acetyl-CoA both of which were present in the crystallization milieu) were well ordered in the active sites of chains A and B and were included in the model. The magnesium ion in the active site of chain C was coordinated by a pyruvate molecule. Additionally, two glycerol molecules and three phosphate ions and five chlorides were located and included in the final model. The coordinates and structure factors have been deposited in the PDB as entry PDB entry 6AQ4.          Figure S10. A proposed mechanism for Rv2498c C-C cleavage of β-hydroxyl-acyl-CoA thioesters. The first step in the reaction is the base catalysed deprotonation of the β-hydroxyl group of HMG-CoA via metal-bound water-mediated proton abstraction. Based on our structures, the general base is either Glu36 or Asp37, both at close proximity (less than 3 angstroms) to the water molecules. This first step generates an alkoxide intermediate. The second step is the C-C bond cleavage as a result of the collapse of the alkoxide resulting in the two reaction products: acetoacetate and acetyl-CoA. The Arg64 is suggested to partially involve in the enolisation of the acetyl-CoA intermediate. The general base is likely regenerated by the metal-bound water-mediated proton abstraction.