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

• Published in: The Journal of biological chemistry Vol. 276; no. 47; pp. 43580 - 43588
• Main Authors: Empadinhas, N, Marugg, J D, Borges, N, Santos, H, da Costa, M S
• Format: Journal Article
• Language: English
• Published: United States 23.11.2001
• Subjects: a-Mannosyl-3-phosphoglycerate; a-Mannosylglycerate; Amino Acid Sequence; Base Sequence; DNA Primers; Glyceric Acids; Kinetics; Mannose - analogs & derivatives; Mannose - biosynthesis; mannosyl-3-phosphoglycerate; mannosyl-3-phosphoglycerate phosphatase; mannosyl-3-phosphoglycerate synthase; mannosylglicerate; Mannosyltransferases - chemistry; Mannosyltransferases - genetics; Mannosyltransferases - metabolism; Molecular Sequence Data; PH0925 gene; PH0926 gene; PH0927 gene; PHO926 gene; PHO927 gene; Phosphotransferases (Alcohol Group Acceptor) - chemistry; Phosphotransferases (Alcohol Group Acceptor) - genetics; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Pyrococcus - enzymology; Pyrococcus - metabolism; Pyrococcus horikoshii; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Substrate Specificity
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M108054200

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.