Pathway for the Synthesis of Mannosylglycerate in the Hyperthermophilic Archaeon Pyrococcus horikoshii

Abstract

The biosynthetic pathway for the synthesis of the compatible solute ␣-mannosylglycerate in the hyperthermophilic archaeon Pyrococcus horikoshii is proposed based on the activities of purified recombinant mannosyl-3-phosphoglycerate (MPG) synthase and mannosyl-3-phosphoglycerate phosphatase. The former activity was purified from cell extracts, and the N-terminal sequence was used to identify the encoding gene in the completely sequenced P. horikoshii genome. This gene, designated PH0927, and a gene immediately downstream (PH0926) were cloned and overexpressed in Escherichia coli. The recombinant product of gene PH0927 catalyzed the synthesis of ␣-mannosyl-3-phosphoglycerate (MPG) from GDP-mannose and D-3-phosphoglycerate retaining the configuration about the anomeric carbon, whereas the recombinant gene product of PH0926 catalyzed the dephosphorylation of mannosyl-3-phosphoglycerate to yield the compatible solute ␣-mannosylglycerate. The MPG synthase and the MPG phosphatase were specific for these substrates. Two genes immediately downstream from mpgs and mpgp were identified as a putative bifunctional phosphomannose isomerase/mannose-1-phosphateguanylyltransferase (PH0925) and as a putative phosphomannose mutase (PH0923). Genes PH0927, PH0926, PH0925, and PH0923 were contained in an operon-like structure, leading to the hypothesis that these genes were under the control of an unknown osmosensing mechanism that would lead to ␣-mannosylglycerate synthesis. Recombinant MPG synthase had a molecular mass of 45,208 Da, a temperature for optimal activity between 90 and 100°C, and a pH optimum between 6.4 and 7.4; the recombinant MPG phosphatase had a molecular mass of 27,958 Da and optimum activity between 95 and 100°C and between pH 5.2 and 6.4. This is the first report of the characterization of MPG synthase and MPG phosphatase and the elucidation of a pathway for the synthesis of mannosylglycerate in an archaeon.

The Bacterium Thermus thermophilus , Like Hyperthermophilic Archaea, Uses a Two-Step Pathway for the Synthesis of Mannosylglycerate

“…The thermophilic bacteria T. thermophilus (classified within the Deinococcus-Thermus phylum) (8), R. marinus (a member of the phylum Cytophaga-Flavobacterium-Bacteroides) (35), and R. xylanophilus (representing the deepest branch within the phylum Acti- (24,25), whose genomes have been sequenced, lack genes for the synthesis of MG (21,27). Two biosynthetic pathways for MG have been identified in R. marinus, but only the two-step pathway has been identified in P. horikoshii (9,26). The identification of the sequences of the genes from P. horikoshii provided us with the information to search for homologs in other organisms that accumulate MG whose genomes have been sequenced and in the unpublished T. thermophilus HB27 genome.…”

“…The reaction mixture was incubated at 70°C for 15 min and cooled on ice, followed by incubation for 15 min with 2 U of alkaline phosphatase (Sigma-Aldrich) at 37°C. The products formed before and after the addition of alkaline phosphatase were visualized by thin-layer chromatography (TLC) (9). Standards of MG, MPG, D-mannose, guanosine, D-3-phosphoglycerate, GDP-mannose, and GDP were used for comparative purposes.…”

“…The solvent system used was composed of n-propanol and ammonia (1:1 by volume), which separated the MPG formed from residual GDP-mannose in the reaction mixture. Visualization of the compounds was achieved with ␣-naphthol-sulfuric acid solution (9).…”

“…The reaction mixtures, containing a 2.5 mM concentration of each substrate in 25 mM Tris-HCl (pH 7.6) and 25 mM MgCl 2 , were incubated at 70°C for 30 min, followed by dephosphorylation at 37°C for 15 min with 2 U of alkaline phosphatase. Samples were tested for the formation of products by TLC (9).…”

“…In the other pathway, mannosyl-3-phosphoglycerate synthase (MPGS) catalyzes the conversion of GDP-mannose and D-3-phosphoglycerate into mannosyl-3-phosphoglycerate (MPG), which is subsequently converted to MG by mannosyl-3-phosphoglycerate phosphatase (MPGP). In contrast to the case in R. marinus, the synthesis of MG in P. horikoshii proceeds only via the two-step pathway involving the phosphorylated intermediate (9).…”