title: 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.
authors.affiliation: Instituto de Tecnologia Quimica e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, Apartado 127, 2780-156 Oeiras, Portugal.
authors.forename: N
authors.initials: N
authors.lastname: Empadinhas
authors.forename: J D
authors.initials: JD
authors.lastname: Marugg
authors.forename: N
authors.initials: N
authors.lastname: Borges
authors.forename: H
authors.initials: H
authors.lastname: Santos
authors.forename: M S
authors.initials: MS
authors.lastname: da Costa
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D005988:Glyceric Acids; D007700:Kinetics; D008358:Mannose / Q000031:analogs & derivatives* / Q000096:biosynthesis*; D008364:Mannosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019711:Pyrococcus / Q000201:enzymology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D005988:Glyceric Acids; D011994:Recombinant Proteins; C402688:mannosylglycerate; D008364:Mannosyltransferases; C441639:mannosyl-3-phosphoglycerate synthase; D017853:Phosphotransferases (Alcohol Group Acceptor); C441640:mannosyl-3-phosphoglycerate phosphatase; D008358:Mannose