Discovery of a Glycerol 3-Phosphate Phosphatase Reveals Glycerophospholipid Polar Head Recycling in Mycobacterium Tuberculosis

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Discovery of a glycerol 3-phosphate phosphatase reveals glycerophospholipid polar head recycling in Mycobacterium tuberculosis

Gérald Larrouy-Maumusa,1, Tapan Biswasb,1, Debbie M. Hunta, Geoff Kellya,c, Oleg V. Tsodikovd,2, and Luiz Pedro Sório de Carvalhoa,2

aDivision of Mycobacterial Research, Medical Research Council National Institute for Medical Research, London NW7 1AA, United Kingdom; bDepartment of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109; cMedical Research Council Biomedical Nuclear Magnetic Resonance Centre, London NW7 1AA, United Kingdom; and dDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536

Edited by Patrick J. Brennan, Colorado State University, Fort Collins, CO, and accepted by the Editorial Board May 31, 2013 (received for review December 11, 2012)

Functional assignment of enzymes encoded by the Mycobacterium in a mycobacterial extract of small molecules acting as a repre- tuberculosis genome is largely incomplete despite recent advances sentative source of physiologically relevant substrates, cofactors, in genomics and bioinformatics. Here, we applied an activity- and allosteric regulators. One of the main advantages of ABMP based metabolomic profiling method to assign function to is the lack of any inherent bias toward the number and type of a unique phosphatase, Rv1692. In contrast to its annotation as substrates or toward the nature of a catalytic mechanism (8). a nucleotide phosphatase, metabolomic profiling and kinetic char- We applied this approach to an M. tuberculosis protein of an acterization indicate that Rv1692 is a D,L-glycerol 3-phosphate unknown function, Rv1692, annotated as a putative nucleotide phosphatase. Crystal structures of Rv1692 reveal a unique archi- phosphatase. Transposon saturation mutagenesis data indicate tecture, a fusion of a predicted haloacid dehalogenase fold with that rv1692 is not essential in vitro (9), but no information regarding is role in vivo is currently available. The enzyme belongs a previously unidentified GCN5-related N-acetyltransferase region. to the haloacid dehalogenase superfamily (HADSF), which Although not directly involved in acetyl transfer, or regulation of contains a large number of enzymes that share a conserved core enzymatic activity in vitro, this GCN5-related N-acetyltransferase domain and yet catalyze diverse reactions (e.g., phosphatase, region is critical for the solubility of the phosphatase. Structural phosphonatase, phosphomutase, and dehalogenase) (10-12). The and biochemical analysis shows that the active site features are HADSF is further divided into three subfamilies, I, II (A and B), adapted for recognition of small polyol phosphates, and not nu- and III, according to the topology and the insertion of the cap cleotide substrates. Functional assignment and metabolomic stud- domain that dictates substrate specificity (10-12). Subfamily I is ies of M. tuberculosis lacking rv1692 demonstrate that Rv1692 is characterized by a small α-helical bundle cap domain located the final enzyme involved in glycerophospholipid recycling/catab- between motifs I and II of the core domain. The subfamily II cap olism, a pathway not previously described in M. tuberculosis. is located between motifs II and III of the core domain and consists of two different α/β folds, designated types IIA and IIB. haloacid dehalogenase superfamily | enzyme function | pathway discovery Contrary to the other two subfamilies, subfamily III does not possess a cap domain and has only a core domain with a con- ach year, 1.4 million people succumb to tuberculosis, making necting loop serving in place of the cap domain (13). Here, we report a detailed functional and structural charac- EMycobacterium tuberculosis the deadliest bacterium affecting fi mankind (1). In addition, the dissemination of strains resistant to terization of Rv1692, revealing its substrate speci city and several antibiotics underscores the need for better understanding unique structural scaffold, as well as providing evidence that of this pathogen and for the development of novel vaccines and implicates it in an unexpected metabolic function in M. tuber- therapeutics (2, 3). Our approach to elucidating the unique per- culosis, the recycling/catabolism of glycerolphospholipid-derived vasiveness of M. tuberculosis is through comprehensive discovery polar heads. and characterization of metabolic pathways, as metabolism underlies survival of the bacteria both inside and outside the host Results and Discussion and can contribute to phenotypic and genetic drug resistance. Rv1692 Is a Unique HADSF Phosphatase. HADSF enzymes contain The M. tuberculosis genome encodes for 4,043 genes, of which a core Rossmann catalytic fold in which highly conserved resi- 3,933 encode proteins (4, 5). Many genes with essential func- dues coordinate a divalent metal. HADSF phosphatases act on tions, such as DNA replication, protein and RNA synthesis, and chemically diverse substrates. Substrate specificity is determined cell-division, have close homologs in other bacteria, and their by a structural insertion in the core, called a cap domain (10-14), functions are annotated largely on the basis of analysis of their counterparts. However, functions of at least one-third of the genes are unknown or putative (4). In particular, little is known Author contributions: G.L.-M., T.B., D.M.H., G.K., O.V.T., and L.P.S.d.C. designed research; about genes that are not conserved or conditionally important. G.L.-M., T.B., D.M.H., and G.K. performed research; L.P.S.d.C. contributed new reagents/ Characterization of such genes presents a daunting task. M. tu- analytic tools; G.L.-M., T.B., D.M.H., G.K., O.V.T., and L.P.S.d.C. analyzed data; and G.L.-M., berculosis is thought to be subjected to a myriad of conditions T.B., O.V.T., and L.P.S.d.C. wrote the paper. during its life cycle in the host, such as low pH, reactive oxygen The authors declare no conflict of interest. and nitrogen species, and so on (6, 7). Understanding how This article is a PNAS Direct Submission. P.J.B. is a guest editor invited by the M. tuberculosis adapts to and even thrives in such diverse envi- Editorial Board. ronments is critical to our understanding of the pathology itself Freely available online through the PNAS open access option. and for the discovery of novel therapeutics to treat tuberculosis. Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, We applied an innovative, activity-based metabolomic profiling www.pdb.org (PDB ID codes 4I9F and 4I9G). (ABMP) approach to assign function to orphan (without a priori 1G.L-M. and T.B. contributed equally to this work. known substrates/products) mycobacterial enzymes and to discover 2To whom correspondence may be addressed. E-mail: [email protected] or oleg. unknown metabolic pathways. ABMP employs liquid chromatog- [email protected]. raphy coupled with accurate time-of-flight mass spectrometry to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. separate, identify, and quantify potential substrates and products 1073/pnas.1221597110/-/DCSupplemental.

11320–11325 | PNAS | July 9, 2013 | vol. 110 | no. 28 www.pnas.org/cgi/doi/10.1073/pnas.1221597110 Downloaded by guest on September 24, 2021 which forms a lid over the active site cavity during catalysis. from Agilent Technologies. Of an initial 5,648 ions detected, only Rv1692 is a potential HADSF member conserved in mycobac- eight ions were identified as changing more than twofold (P < 0.01; teria. In addition to a clearly identifiable HADSF core, Rv1692 SI Appendix, Fig. S3). Subsequent analysis indicated that all eight contains a weakly conserved cap domain that includes putative are false-positives. substrate specificity determinants (SI Appendix, Fig. S1), as in- As peak recognition and retention time variations are likely to dicated by sequence homology analysis. The closest character- mask positive results obtained by automated methods, we carried ized homolog of Rv1692 is NagD from Escherichia coli (28% out manual inspection of all mass spectral data obtained in the identity of the HADSF core residues), a nucleotide phosphatase experiments in both positive and negative ionization modes. specific to UMP and GMP. However, unlike NagD, Rv1692 Indeed, manual inspection of the data indicated that one ion − contains an extra 103-residue C-terminal region in addition to with m/z 171.0067 [M-H] disappeared completely on addition of the core and cap domains (SI Appendix, Fig. S1). This region Rv1692 (Fig. 1). The extracted ion chromatogram corresponding does not have an identifiable homolog. Phylogenetic analysis to this m/z did not display a single peak but, instead, displayed using Basic Local Alignment Search Tool (BLAST) and Mo- a set of peaks, which is characteristic of phosphorylated small + lecular Evolutionary Genetic Analysis (MEGA5) (15, 16) indi- molecules and their adducts with Na ion inter alia (Fig. 1A). cates that the Rv1692 homologs that contain this extension of the Early elution time was consistent with acidic or phosphorylated HADSF core are confined to the Actinomycetales phylum (high compounds. A single empirical formula, C H O P, was obtained − 3 5 6 GC content, Gram-positive bacteria). Interestingly, Rv1692 homo- for the m/z of 171.0067 [M-H] . The most likely metabolite from logs from most pathogenic and opportunistic mycobacterial known bacterial metabolic pathways that matches this formula is species such as M. tuberculosis, Mycobacterium ulcerans,and glycerol phosphate (theoretical m/z 171.0064, Δppm = 1.75). LC- Mycobacterium marinum are found in group I (SI Appendix, Fig. MS assays with synthetic D,L-glycerol 3-phosphate (G3P) are S2 and Table S1), whereas most of the nonpathogenic homologs consistent with this conclusion (SI Appendix, Fig. S4). Therefore, are found in group II, suggesting a possible role in disease. It is our metabolomic data suggest that Rv1692 breaks down glycerol noteworthy that genes encoding additional NagD homologs phosphate into glycerol and inorganic phosphate. It is important lacking this extension are present in some species, suggesting to stress that other phosphorylated metabolites detectable in our a divergent function of Rv1692. LC-MS–based assay such as hexose-phosphate, dihydroxyacetone phosphate/glyceraldehydes 3-phosphate, and glycerophospholipids Search for Potential Substrates of Rv1692 by Activity-Based Meta- polar heads did not show changes in absence or presence of bolomic Profiling. We cloned, expressed in E. coli, and purified Rv1692 in a time-dependant manner (SI Appendix,Fig.S5). Rv1692 (SI Appendix, Materials and Methods). Phosphatase activity assays indicate that despite its homology to NagD, UMP and Rv1692 Is a G3P Phosphatase. To directly investigate the substrate GMP are not substrates of Rv1692 (SI Appendix,TableS2). This specificity of Rv1692, steady-state kinetic measurements were observation, together with the presence of an extra domain and performed using a variety of substrates. In total, we tested 32 a differentially evolved cap domain in Rv1692, suggest that phosphorylated metabolites, mainly from established metabolic Rv1692 has a distinct substrate specificity and function. To search pathways, including chemical analogs of glycerol phosphate (e.g., for a substrate of Rv1692, we applied ABMP (8). In this method, linear compounds, cyclic sugars and nucleotides, phosphorylated we prepared an extract of polar metabolites from mycobacteria coenzymes, and phosphorylated amino acids; Table 1 and SI (17) and used it as a library of potential in vitro substrates. This Appendix, Table S2). Among all the substrates tested, G3P, small-molecule extract (SME) contains ∼1,700 unique ions (8). glycerol 2-phosphate, ribulose 5-phosphate, L-glycerol 3-phos- The identity and the abundance of ionizable polar small molecules phate, and a nonphysiological substrate para-nitrophenyl phos- in the SME are determined in both the absence and the presence phate (pNPP) exhibited measurable activity (Table 1). Rv1692 of the purified Rv1692 and are analyzed in both positive and preferentially catalyzed the hydrolysis of G3P (V/K = 0.86 × 103 − − m negative ion modes. Substrates and products are identified on the M 1·s 1). This enzyme was also able to act on glycerol 2-phos- 3 −1 −1 basis of their accurate mass (<5 ppm), retention time, and isotopic phate (V/Km = 0.14 × 10 M ·s ) and D-ribulose 5-phosphate + 3 −1 −1 envelope. The ion with m/z = 325.0431 [M+H] , which corresponds (V/Km = 0.14 × 10 M ·s ), albeit with significantly lower ef- to UMP, did not show significant changes as a function of time and ficiencies. pNPP was also hydrolyzed with a drastically lower 3 −1 −1 presence of Rv1692, which agrees with the lack of phosphatase efficiency (V/Km = 0.05 × 10 M ·s ; Table 1). Interestingly, we activity with UMP (SI Appendix,TableS2). Unsupervised statistical did not observe any activity toward D-glyceraldehyde 3-phosphate analysis of the data was carried using Mass Profiler Professional, or dihydroxyacetone-phosphate, which are metabolites closely

Fig. 1. Activity-based metabolomic proﬁling reveals BIOCHEMISTRY glycerol phosphate phosphatase activity of Rv1692. Metabolomic proﬁling of SME was carried out in the presence (red trace) or absence (black trace) of Rv1692 in negative ion mode. (A and B) Extract ion chromatograms in the absence and presence of Rv1692, respectively. (Insets C and D) Region of in- terested of the mass spectrum (from the retention time of 1–2.5 min). The green box was added to fa- cilitate visualization of the correct ion, which is not present on Inset D. In the absence of Rv1692, we observe an ion with m/z 171.0067, which is within a 1.75-ppm error of the theoretical expected value for glycerol phosphate (m/z 171.0064). In the presence of Rv1692, the 171.0067 ion is completely con- sumed. The ion with m/z 171.0103 is within a 22.8- ppm error of 171.0064; therefore, it represents another compound. The data are representative of two independent experiments.

Larrouy-Maumus et al. PNAS | July 9, 2013 | vol. 110 | no. 28 | 11321 Downloaded by guest on September 24, 2021 Table 1. Binding and steady-state kinetic parameters for Rv1692 −1 −1 −1 3 Substrates Kd, μM* kcat,s Km, mM Hill no. kcat/Km,s ·M (×10 )

G3P 165.3 ± 22.3 0.77 ± 0.03 0.89 ± 0.01 2 0.86 ± 0.08 D-ribulose 5-phosphate 201.8 ± 35.1 0.18 ± 0.05 1.30 ± 0.40 2 0.14 ± 0.06 Glycerol 2-phosphate 328.7 ± 23.6 0.18 ± 0.01 2.01 ± 0.18 2 0.09 ± 0.01 Para-nitrophenyl phosphate 60.8 ± 2.7 1.00 ± 0.20 19.30 ± 5.30 1 0.05 ± 0.02 L-glycerol 3-phosphate 158.2 ± 22.2 0.02 ± 0.01 1.10 ± 0.20 1 0.02 ± 0.01

Assays were performed in Tris·HCl 50 mM 1mM MgCl2 at pH 7.5, using the EnzCheck phosphate detection kit. *Kd measurements were performed by determining the quenching of intrinsic tryptophan ﬂuorescence, with 1 μM Rv1692 in buffer Tris HCl 50 mM at pH 7.5.

+ related to glycerol 3-phosphate. This signifies either that both Mn2+ and Mg2+, respectively). For Ni2 , the catalytic efficiency − − hydroxyl groups of glycerol 3-phosphate are required for binding (V/K = 1.25 × 103 M 1·s 1) is approximately half of that for +act or that sp2 hybridized substrates are not accommodated at the Mg2 . Higher concentrations of divalent metals lead to enzyme active site. In addition, we observed that the stereochemistry was inhibition (SI Appendix, Table S3). Although the specificity for 2+ 2+ also critical for activity. Even though the values of Km obtained Mg is eight- and fourfold fold lower than those for Co and 2+ 2+ for both L-glycerol 3-phosphate and G3P are the same within Mn , respectively, Mg is likely the physiologically relevant experimental error, at 0.89 ± 0.1 mM and 1.10 ± 0.20 mM, re- catalytic divalent metal ion. The free intracellular concentra- 2+ spectively, the turnover rate constant (kcat) for G3P (0.77 ± 0.03 tion of Mg in E. coli is ∼1–2 mM (21), which is similar to the −1 2+ 2+ s ) is 40-fold higher than that for L-glycerol 3-phosphate (0.02 ± K value for Mg , whereas intracellular concentrations for Mn − act + 0.01 s 1). Therefore, Rv1692 is a glycerol-phosphate phosphatase (0.01 μM; ref. 22) and Co2 (0.001 μM; ref. 23) are considerably with a preference for D-glycerol 3-phosphate over L-glycerol smaller than the corresponding Kact values. 3-phosphate. Altogether, these data indicate that the substrate-binding A GNAT-Like Fused Region Is Required for Rv1692 Solubility. To pocket of Rv1692 can efficiently accommodate small linear clarify the role of the C-terminal region in the activity of Rv1692, + + carbon backbones, containing two free hydroxyl groups, but not we crystallized Rv1692 with Mg2 and Ca2 and determined cyclic sugars or nucleotides. The V/K value for G3P (∼103 these two structures (SI Appendix, Table S4). The crystal struc- − − m − − M 1·s 1) is below the threshold of 104 M 1·s 1 expected for in- tures show that Rv1692 contains a HADSF fold and forms a termediate metabolism enzymes, suggesting a function outside dimer similarly to other HADSF phosphatases (10). The core metabolism. Until now, only two D-glycerol 3-phosphate C-terminal extension of Rv1692, which is absent in other char- phosphatase activities from bacteria have been reported: one acterized HADSF members, resembles a small GCN5-related from Bacillus licheniformis (18) and the other from Co- N-acetyltransferase (GNAT) fold (24) (Fig. 2). This region is rynebacterium glutamicum (19). Regarding the latter, Linder fused to the HADSF catalytic domain and does not appear to be et al. found that the G3P phosphatase deletion mutant strain an independent domain. To check for an allosteric effect of this showed a significantly reduced growth rate but had a final bio- domain on the phosphatase activity, we measured the phos- mass comparable to the wild-type strain when cultured in a me- phatase activity of Rv1692 with G3P as substrate in the presence dium containing 40 g/L glucose and 1 mM G3P as carbon or absence of typical GNAT cosubstrates acetyl-CoA (AcCoA) sources. These data are in accordance with the inability of the and succinyl-CoA (SucCoA) or their product, CoA. (SI Ap- mutant strain to rapidly metabolize G3P. The authors of that pendix,TableS5). We also tested whether or not this fused study concluded that this G3P phosphatase activity may function GNAT domain possessed acetyltransferase or hydrolase activi- to prevent the intracellular accumulation of G3P under meta- ties. No effect on the phosphatase activity and no acetyl- or bolically unbalanced conditions. succinyltransferase or hydrolase activities were detected (SI Ap- pendix, Table S6). This was further confirmed by 1H-NMR, in Divalent Metal Ion Specificity of Rv1692. HADSF phosphatases which no formation of acetyl-glycerol or generation of CoA was + + require divalent metals, usually Mg2 or Mn2 , for activity (20). observed (SI Appendix, Fig. S6). Altogether, these results indicate To probe the specificity of Rv1692 toward divalent metals, we that this GNAT region is not likely to be involved in acetyl group performed steady-state kinetic measurements of hydrolysis of transfer using AcCoA and SucCoA, it could nonetheless be pNPP as a function of concentration of the divalent metal. The a regulatory domain with a hitherto unknown mechanism kinetic parameters are presented in SI Appendix, Table S3. of action. + + + Among the divalent metals tested, Mg2 ,Co2 ,Mn2 ,and Finally, we generated two Rv1692 truncation mutants lacking + + + Ni2 supported catalysis. Co2 and Mn2 support eight- and the GNAT region to assess its role in activity and/or structural 2+ fourfold higher catalytic efficiency than Mg (V/K = 27.20 × stability. The truncated variants (Rv1692 – or Rv1692 – ) − − − − − act− 1 265 1 267 103 M 1· s 1,13.27× 103 M 1·s 1,and3.27× 103 M 1·s 1 for Co2+, were well expressed in E. coli but were insoluble (SI Appendix,

Fig. 2. Crystal structure of Rv1692. (A) Dimerization of Rv1692 is enabled through the cap domains of the monomers. The HADSF core, cap, and fused GNAT regions are highlighted in orange, gray, and green, respectively. (B) A 90° rotated view of the dimer of Rv1692 (C) A cartoon representation of a monomer showing the C-terminal GNAT region fused to the HADSF core.

11322 | www.pnas.org/cgi/doi/10.1073/pnas.1221597110 Larrouy-Maumus et al. Downloaded by guest on September 24, 2021 Fig. S7), indicating that the GNAT region is required for the solubility of the HADSF fold of Rv1692 and is potentially needed for the structural integrity of this enzyme. Indeed, deletion of the GNAT region exposes several hydrophobic residues of Rv1692 to solvent (SI Appendix, Fig. S1), which likely causes aggregation of the deletion mutants and may even cause their misfolding. Rv1692 is the ﬁrst member of HADSF that contains an integral GNAT-like region, thus deﬁning a new subfamily, subfamily C, belonging to the HADSF II group.

The Active Site and Substrate Binding. Several crystal structures of NagD-like HADSF enzymes are available, but only a few of them contain bound substrates; for example, a predicted phosphatase from Saccharomyces cerevisiae with glycerol 3-phosphate bound (PDB code 3RF6), human pyridoxal 5′-phosphate phosphatase with pyridoxal 5′-phosphate bound (PDB code 2CFT), and human phospholysine/phosphohistidine phosphatase in complex with 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (PDB code 2X4D). In these structures, the cap domain is in a closed conformation. From comparing our structures of Rv1692 with these and other HADSF structures, it can be observed that the substrate binding pockets of both Rv1692 structures are in open conformation (SI Appendix, Fig. S8 A–C). We modeled the cap domain in the closed conformation to analyze the shape of the pocket and the substrate-interacting surface (SI Appendix, Fig. S8 D–F). The closed conformation of the cap of Rv1692 was modeled on the basis of the structures of the human pyridoxal 5′ phosphate phosphatase and the predicted phosphatase from S. cerevisiae. Because the conserved residues of the catalytic do- + main involved in Mg2 -scissile phosphate coordination are po- sitioned nearly identically, they serve as reference, making such modeling highly predictive. In the model, the substrate binding pocket closely resembles that of the S. cerevisae phosphatase, where the cap domain provides a very similar surface for binding Fig. 3. Substrate binding pocket of Rv1692. (A) The modeled pocket in to glycerol 3-phosphate (Fig. 3A) and cannot accommodate closed conformation with modeled glycerol 3-phosphate (G3P; ligand from – PDB code 3RF6). (B–D) The pocket with modeled adenosine 5′phosphate larger substrates such as nucleotides or sugars (Fig. 3 B D). In ′ particular, residues I156 and P157 must contact the bound sub- (AMP; from PDB code 3OCV), uridine 5 -phosphate (U5P; from PDB code 3OPX), and 1,7-di-O-phosphono-L-glycero-β-D-manno-heptopyranose (GMB; strate similarly to residues W209 and A210 of the S. cerevisae fi from PDB code 3L8G), respectively, showing clash of the ligands with Rv1692 protein. In agreement with substrate speci city studies, larger active site residues. sugar phosphates and nucleosides cannot be accommodated in the small active site of Rv1692. compared with the wild-type strain, suggesting that glycerol M. tuberculosis Deletion of rv1692 in Results in an Accumulation of derived from Rv1692 is unlikely to significantly affect the level G3P and G3P-Containing Lipid Polar Heads. To examine the G3P fi of gluconeogenesis. phosphatase activity of Rv1692 in vivo and to de ne its biological According to these results and the kinetic parameters, Rv1692 role, an in-frame, unmarked deletion mutant of rv1692 was Δ does not appear to be a core metabolic enzyme but, rather, is constructed in M. tuberculosis H37Rv ( rv1692)(SI Appendix, involved in the recycling/catabolism of glycerophospholipid polar Fig. S9). LC-MS analysis of the metabolome of wild-type, Δ heads. To provide independent evidence for lipid polar head rv1692, and complemented strains was performed as previously recycling/catabolism in M. tuberculosis, we performed experi- described (8, 25). Targeted metabolomic analysis shows an in- 13 ments using U- C glycerol as sole carbon source and followed crease of the pool size of glycerol phosphate in the Δrv1692 the incorporation of the label in the wild-type, Δrv1692, and compared with the wild-type (Fig. 4). The pool size of reference metabolites such as aspartic acid, glutamine, histidine, and serine complemented strains. An increase in labeling could be expected remain unchanged (Fig. 4). This result is in accordance with the if Rv1692 was involved in the catabolism of lipid polar heads, BIOCHEMISTRY glycerol phosphate phosphatase activity assigned to Rv1692. and a decrease in labeling could be expected if Rv1692 was involved in intermediate metabolism, decreasing the flux of glyc- Genetic complementation restored the glycerol phosphate pool fi to wild-type levels (Fig. 4), confirming that the effect on glycerol erol phosphate to glycolysis and gluconeogenesis. A signi cant phosphate pool size is not a result of a polar effect on a down- increase in the labeling of glycerophospho-ethanolamine and Δ stream gene or other experimental artifacts. Notably, the level of glycerolphospho-inositol is observed in rv1692 compared with specific lipid polar heads containing glycerol phosphate was also wild-type and complemented strains (SI Appendix,Fig.S10). perturbed in the deletion mutant. Glycerophosphoinositol and This result is fully consistent with a decrease in the rate of phosphatidylglycerol levels increased twofold or more, but glyc- degradation/catabolism of these lipid polar heads in the absence erophosphoethanolamine level remained unchanged (Fig. 4). All of Rv1692. three of these polar heads are found in M. tuberculosis’s glycer- Altogether, the results presented here strongly suggest that ophospholipids (26). It is noteworthy that these lipid polar heads Rv1692 is involved in glycerophospholipid polar heads recycling/ are not biosynthetic intermediates in glycerophospholipogenesis, catabolism at the last step (Fig. 5). Such pathways have been and therefore might be indicative of a glycerophospholipid cat- partially characterized in E. coli lysates by Albright et al. (27). In abolic pathway. Importantly, these glycerol-phosphate-contain- this study, all enzymatic activities necessary for glycerophospholipid ing polar heads are not substrates of Rv1692, as no changes were polar head recycling were identified in protein lysates. It was also observed during ABMP. Also worth noting is that the amount found that polar heads were obtained from phospholipids after of hexose phosphate remained unchanged in the Δrv1692 action of phospholipases A1 and A2, which can be localized in the

Larrouy-Maumus et al. PNAS | July 9, 2013 | vol. 110 | no. 28 | 11323 Downloaded by guest on September 24, 2021 heads (Fig. 5). It is important to stress that this catabolic pathway has neither been described in M. tuberculosis before nor predicted based on genomic information, despite the fact that some of the enzymes from this pathway in other organisms have de- fined homologs in M. tuberculosis (Fig. 5). In addition to the evidence presented in this study and the identification of homologs of all enzymes required for lipid polar head catabolism, a few studies support its relevance in vivo. Fatty acid catabolism is well documented in M. tuberculosis. Glycerophospholipid catabolism provides lipid polar heads and fatty acids, which could be degraded through β-oxidation, generating energy and biosynthetic precursors via Krebs and glyoxylate cycles and gluconeogenesis (32–35). Glycerophospholipid turnover has also been verified in related species, such as Mycobacterium smegmatis and Mycobacterium phlei (36). Finally, Rv3842 is essential in vivo during macrophage infection (37). Rv3842 encodes the phosphodiesterase, which breaks down lipid polar heads, releasing glycerol phosphate (37). In conclusion, by using a combination of metabolomic profiling, enzymology, genetics, and structural studies, we established Rv1692 as the first G3P phosphatase from M. tuberculosis. The enzyme contains a GNAT region fused to the HADSF fold, which is essential for structural integrity. This phosphatase is likely involved in the recycling of glycerol phosphate-containing lipid polar heads, catalyzing the last predicted reaction of this Δ pathway in M. tuberculosis. This additional feature is essential for Fig. 4. Targeted metabolomic analysis of M. tuberculosis rv1692 reveals structural integrity. Homologs of Rv1692 are found in other glycerophospho polar head accumulation. Accumulation of glycerol-phosphate and glycerol-phosphate-containing polar heads in M. tuberculosis Δrv1692 is Actinomycetales, suggesting that this function is not restricted to consistent with the functional assignment. The abundances of representative mycobacteria. Further studies will determine the role of this metabolites from the central-carbon metabolism and polar head catabolism recycling pathway in M. tuberculosis during growth in vitro, in are presented as ratios of Δrv1692/wild-type and complemented/wild-type. The macrophages, and in animal models of infection. data are representative of two independent experiments. Materials and Methods Materials. All chemicals were purchased from Sigma. Chromatographic col- wall, inner membrane, and cytosol. In this last compartment, these umns were purchased from GE Healthcare. EDTA-free protease inhibitor was authors found a phosphodiesterase activity that converted phos- phoethanolamine into ethanolamine and glycerol 3-phosphate. Such phospholipase activities have been identified in M. tuberculosis. Schué et al. (28) isolated two secreted cutinases-like proteins: Rv1984c, which preferentially hydrolyzed medium-chain carboxylic esters and monoacylglycerols, and Rv3452, which behaved like a phospholipase A2. The other lipases/cutinases are most likely cell-wall-associated and surface-exposed (29). Concerning the subsequent steps in recycling of phospholipids, in the M. tuberculosis genome, Rv0317c (glpQ2) and Rv3842c (glpQ1) are annotated as glycerophosphoryl diester phosphodiesterases that may degrade polar heads generating glycerol 3-phosphate and the corresponding alcohols. It is interesting to point out that G3P itself might induce accumulation of these lipid polar heads by direct inhibition of the phosphodiesterases. G3P levels in M. tuberculosis are known to vary dramatically, depending on the carbon source used (25), and therefore the nutritional status of the cell might directly regulate glycerophospholipid catabolism via inhibition of GlpQ phosphodiesterases. These activities and their potential inhibition by G3P remain to be confirmed experimentally. G3P can then follow three paths. In the first path, G3P can be acylated by Rv1551 and then by Rv2182c, Rv2482c to generate 1,2-diacyl-sn-glycerol 3-phosphate leading to phospholipids after addition of the polar heads. In the second path, G3P can be directed for glycolysis and gluconeogenesis. In the third path, described here, G3P is broken down by Rv1692 to generate glycerol and inorganic phosphate. Glycerol can leave the cell by passive or facilitated diffusion, serve as an osmolyte, or be used Fig. 5. Schematic representation of a potential polar head recycling pathway used in M. tuberculosis. On enzymatic action of secreted or cell wall- as a central building block in secondary metabolite or lipid an- associated cutinases/lipases, the phospholipids polar heads are released. tigen biosynthesis. For example, Layre and colleagues described These polar heads are subsequently cleaved by glycerophosphoryl diester the discovery of a glycerol-based lipid antigen, glycerol mono- phosphodiesterases (Rv0317c and Rv3842c) generating G3P. G3P can then be mycolate, in M. tuberculosis (30). In addition, diarabinosyl glyc- acylated by Rv1551, followed by Rv2182c and Rv2482c, to form 1,2-diacyl-sn- erol dimycolate has also been described in M. tuberculosis (31). glycerol 3-phosphate, committing it to phospholipids biosynthesis. Alterna- These observations strongly suggest that Rv1692 may function in tively, G3P can be cleaved by Rv1692, generating glycerol and inorganic recycling/catabolism of glycerol and inorganic phosphate, thus phosphate. Glycerol could function as an osmolyte, used in secondary me- acting in the last step of catabolism of glycerophospholipid polar tabolite production, or leave the cell by passive or facilitative diffusion.

11324 | www.pnas.org/cgi/doi/10.1073/pnas.1221597110 Larrouy-Maumus et al. Downloaded by guest on September 24, 2021 purchased from Roche. BL21(DE3) pLysS cells and Ni-NTA resin were pur- ACN mixtures. After centrifugation at 20,000 × g for 10 min at 4 °C, samples chased from EMD. were stored at −80 °C until analyzed by LC-MS, as described in SI Appendix, Materials and Methods. Bioinformatic Analysis. The primary sequence alignment was performed with ClustalW (15). Tree topography and the evolutionary distance are mapped Bacterial Strains and Cultivation. Construction of rv1692-deleted and com- by the neighbor-joining method and constructed using MEGA5 (16). plemented M. tuberculosis H37Rv strains was performed using a suicide plasmid method (38) (SI Appendix, Materials and Methods). Analysis of the Preparation of SME for ABMP. SME was prepared as described earlier (8). metabolites obtained from these strains was performed as described by de Briefly, the Mycobacterium bovis bacillus Calmette–Guérin pellet was sus- Carvalho et al. (25) (SI Appendix, Materials and Methods). pended in 10 mL acidic ACN solution (acetonitrile, 0.2% acetic acid), and cells were disrupted by sonication. Soluble extract was obtained by centrifuga- ACKNOWLEDGMENTS. We thank Dr. Steve Howell for Electrospray ionization at 20,000 × g for 10 min at 4 °C and then flash-frozen and lyophilized. tion-MS analysis of purified Rv1692 and the Large Scale Laboratory for E. coli Lyophilized SME was suspended in 20 mM Tris HCl at pH 7.4, and insoluble growth. We also thank Dr. Kathryn E. A. Lougheed for the pTetR3 plasmid, material was removed by centrifugation. Aliquots were stored at −80 °C. Elena Kondrashkina and the staff of sector LS-CAT (Advanced Photon Source, Argonne National Laboratory) for assistance with the collection of μ the diffraction data, Dr. Yuri Wolf for preliminary phylogenetic analysis of ABMP. Samples (typically 20 L) of the SME were incubated for various the rv1692 gene, and Prof. Vern Schramm for careful reading of the manu- μ lengths of time in the presence or absence of Rv1692 (5 M) and MgCl2 script. This work was supported by funds from the Medical Research Council (4 mM) in a final volume of 250 μL 20 mM Tris·HCl at pH 7.5. Cold ACN (MC_UP_A253_1111; to L.P.S.d.C.) and start-up funds from the University of containing 0.2% acetic acid was used for quenching, yielding 70% (vol/vol) Michigan College of Pharmacy (to O.V.T.).

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