Pathway for the Synthesis of Mannosylglycerate in the Hyperthermophilic Archaeon Pyrococcus horikoshii: BIOCHEMICAL AND GENETIC CHARACTERIZATION OF KEY ENZYMES

Abstract

The biosynthetic pathway for the synthesis of the compatible solute alpha-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 alpha-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 alpha-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-phosphate-guanylyltransferase (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 alpha-mannosylglycerate synthesis. Recombinant MPG synthase had a molecular mass of 45,208 Da, a temperature for optimal activity between 90 and 100 degrees 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 degrees 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.