Pathway for the synthesis of mannosylglycerate in the hyperthermophilic archaeon Pyrococcus horikoshii - Biochemical and genetic characterization of key enzymes

The biosynthetic pathway for the synthesis of the compatible solute alpha - mannosylglycerate in the hyperthermophilic archaeon Pyrococcus horikoshii i s proposed based on the activities of purified recombinant mannosyl-3-phosp hoglycerate (MPG) synthase and mannosyl-3-phosphoglycerate phosphatase. The former activity was purified from cell extracts, and the N-terminal sequen ce was used to identify the encoding gene in the completely sequenced P. ho rikoshii genome. This gene, designated PH0927, and a gene immediately downs tream (PH0926) were cloned and overexpressed in Escherichia coli. The recom binant product of gene PH0927 catalyzed the synthesis of alpha -mannosyl-be ta -phosphoglycerate (MPG) from GDP-mannose and D-3-phosphoglycerate retain ing the configuration about the anomeric carbon, whereas the recombinant ge ne product of PH0926 catalyzed the dephosphorylation of mannosyl-3-phosphog lycerate to yield the compatible solute a-mannosylglycerate. The MPG syntha se and the MPG phosphatase were specific for these substrates. Two genes im mediately downstream from mpgs and mpgp were identified as a putative bifun ctional phosphomannose isomeraselmannose-1-phosphateguanylyltransferase (PH 0925) and as a putative phosphomannose mutase (PH0923). Genes PH0927, PH092 6, PH0925, and PH0923 were contained in an operon-like structure, leading t o the hypothesis that these genes were under the control of an unknown osmo sensing mechanism that would lead to a-mannosylglycerate synthesis. Recombi nant MPG synthase had a molecular mass of 45,208 Da, a temperature for opti mal activity between 90 and 100 degreesC, 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 degreesC and between pH 5.2 and 6.4. Th is is the first report of the characterization of MPG synthase and MPG phos phatase and the elucidation of a pathway for the synthesis of mannosylglyce rate in an archaeon.