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Papers In Press, published online ahead of print September 18, 2001
Instituto de Tecnologia Quimica e Biologica, Oeiras 2780-156 Oeiras
Corresponding Author: santos{at}itqb.unl.pt
The biosynthetic pathway for the synthesis of the compatible solute
J. Biol. Chem, 10.1074/jbc.M108054200
Submitted on August 21, 2001
Revised on September 18, 2001
Accepted on September 18, 2001
Pathway for the synthesis of mannosylglycerate in the hyperthermophilic Archaeon Pyrococcus horikoshii: Biochemical and genetic characterization of key-enzymes
-mannosylglycerate (MG) in the hyperthermophilic archaeon Pyrococcus horikoshii is proposed based on the activities of purified recombinant mannosyl-3-phosphoglycerate synthase (MPG synthase) and mannosyl-3-phosphoglycerate phosphatase (MPG phosphatase). The former activity was purified from cell extracts and the N-terminus sequence 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, while 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-phosphate guanylyl transferase (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 MG synthesis. Recombinant MPG synthase had a molecular mass of 45,208 Da, a temperature for optimal activity between 90 and 100ºC 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 100ºC 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.
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