Mycobacterium tuberculosis Rv2419c, the missing glucosyl-3-phosphoglycerate phosphatase for the second step in methylglucose lipopolysaccharide biosynthesis

Abstract

Mycobacteria synthesize intracellular methylglucose lipopolysaccharides (MGLP) proposed to regulate fatty acid synthesis. Although their structures have been elucidated, the identity of most biosynthetic genes remains unknown. The first step in MGLP biosynthesis is catalyzed by a glucosyl-3-phosphoglycerate synthase (GpgS, Rv1208 in Mycobacterium tuberculosis H37Rv). However, a typical glucosyl-3-phosphoglycerate phosphatase (GpgP, EC3.1.3.70) for dephosphorylation of glucosyl-3-phosphoglycerate to glucosylglycerate, was absent from mycobacterial genomes. We purified the native GpgP from Mycobacterium vanbaalenii and identified the corresponding gene deduced from amino acid sequences by mass spectrometry. The M. tuberculosis ortholog (Rv2419c), annotated as a putative phosphoglycerate mutase (PGM, EC5.4.2.1), was expressed and functionally characterized as a new GpgP. Regardless of the high specificity for glucosyl-3-phosphoglycerate, the mycobacterial GpgP is not a sequence homolog of known isofunctional GpgPs. The assignment of a new function in M. tuberculosis genome expands our understanding of this organism's genetic repertoire and of the early events in MGLP biosynthesis.

Figure 1. Proposed pathway for the synthesis of the MGLP in M. tuberculosis.

Confirmed activities are shaded in blue. White boxes indicate putative/deduced enzyme activities. Genes linked to the MGLP pathway by mutagenesis studies are indicated in red. GpgS, glucosyl-3-phosphoglycerate synthase; GpgP, glucosyl-3-phosphoglycerate phosphatase; DggS, di-glucosylglycerate synthase; GT, glucosyltransferases; MeTr, methyltransferases; AcTr, acyltransferases.

Figure 2. Flow diagrams of the purification processes for the native and recombinant GpgPs.

(A) Purification of the native GpgP from M. vanbaalenii and SDS-PAGE gel with the purest fractions obtained. (B) Purification from E. coli extracts of the M. tuberculosis recombinant GpgP and SDS-PAGE gel with the pure recombinant enzyme (three consecutive fractions eluting from the final step).

Figure 3. Genetic context of the gpgP genes in mycobacteria and closely related actinobacteria.

gpgP – glucosyl-3-phosphoglycerate phosphatase gene. nadD – probable nicotinate-nucleotide adenylyltransferase gene. orf, open reading frame of unknown function. Amino acid identity between GpgP homologues is indicated.

Figure 4. (A) Unrooted phylogenetic tree based on the amino acid sequences of identified and putative GpgPs/MpgPs (EC 3.1.3.70), PGMs (EC 5.4.2.1) and of other enzymes of the histidine phosphatase superfamily.

Organisms where GG (or MG) has been detected are shaded in yellow. Organisms with enzymes shown to dephosphorylate GPG (or MPG) are boxed. E. coli enzymes with confirmed function are highlighted in blue. The mycobacterial GpgPs studied in this work are highlighted in red. Peptide accession numbers (NCBI) are indicated. Scale bar, 0.2 changes per site. (B) Alignment of the N-terminal amino acid sequences of the PGM from E. coli, the GpgPs from M. vanbaalenii (Mvan_3924) and from M. tuberculosis (Rv2419c) and its paralogs. The typical “RHG” motif of the histidine phosphatase superfamily is boxed.

Figure 5. (A) Substrate preference of the recombinant GpgP from M. tuberculosis, (B) Temperature profile and (C) pH dependence.

GPG, MPG, MGPG and pNPP were tested as substrates for dephosphorylation; 3-PGA and 2-PGA were tested as substrates for PGM activity.