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

Abstract

Functional assignment of enzymes encoded by the Mycobacterium tuberculosis genome is largely incomplete despite recent advances in genomics and bioinformatics. Here, we applied an activity-based metabolomic profiling method to assign function to a unique phosphatase, Rv1692. In contrast to its annotation as a nucleotide phosphatase, metabolomic profiling and kinetic characterization indicate that Rv1692 is a D,L-glycerol 3-phosphate phosphatase. Crystal structures of Rv1692 reveal a unique architecture, a fusion of a predicted haloacid dehalogenase fold with a previously unidentified GCN5-related N-acetyltransferase region. Although not directly involved in acetyl transfer, or regulation of enzymatic activity in vitro, this GCN5-related N-acetyltransferase region is critical for the solubility of the phosphatase. Structural and biochemical analysis shows that the active site features are adapted for recognition of small polyol phosphates, and not nucleotide substrates. Functional assignment and metabolomic studies of M. tuberculosis lacking rv1692 demonstrate that Rv1692 is the final enzyme involved in glycerophospholipid recycling/catabolism, a pathway not previously described in M. tuberculosis.

Keywords: enzyme function; haloacid dehalogenase superfamily; pathway discovery.

Fig. 1.

Activity-based metabolomic profiling reveals 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 interested of the mass spectrum (from the retention time of 1–2.5 min). The green box was added to facilitate 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 consumed. 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.

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.

Fig. 3.

Substrate binding pocket of Rv1692. (A) The modeled pocket in closed conformation with modeled glycerol 3-phosphate (G3P; ligand from PDB code 3RF6). (B–D) The pocket with modeled adenosine 5′phosphate (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; from PDB code 3L8G), respectively, showing clash of the ligands with Rv1692 active site residues.

Fig. 4.

Targeted metabolomic analysis of M. tuberculosis Δrv1692 reveals glycerophospho polar head accumulation. Accumulation of glycerol-phosphate and glycerol-phosphate-containing polar heads in M. tuberculosis Δrv1692 is consistent with the functional assignment. The abundances of representative metabolites from the central-carbon metabolism and polar head catabolism are presented as ratios of Δrv1692/wild-type and complemented/wild-type. The data are representative of two independent experiments.

Fig. 5.

Schematic representation of a potential polar head recycling pathway used in M. tuberculosis. On enzymatic action of secreted or cell wall-associated cutinases/lipases, the phospholipids polar heads are released. These polar heads are subsequently cleaved by glycerophosphoryl diester phosphodiesterases (Rv0317c and Rv3842c) generating G3P. G3P can then be acylated by Rv1551, followed by Rv2182c and Rv2482c, to form 1,2-diacyl-sn-glycerol 3-phosphate, committing it to phospholipids biosynthesis. Alternatively, G3P can be cleaved by Rv1692, generating glycerol and inorganic phosphate. Glycerol could function as an osmolyte, used in secondary metabolite production, or leave the cell by passive or facilitative diffusion.