The bacterium Thermus thermophilus, like hyperthermophilic archaea, uses a two-step pathway for the synthesis of mannosylglycerate.
The biosynthetic pathway for the synthesis of the compatible solute alpha-mannosylglycerate (MG) in the thermophilic bacterium Thermus thermophilus HB27 was identified based on the activities of recombinant mannosyl-3-phosphoglycerate synthase (MPGS) (EC 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (MPGP) (EC 3.1.3.70). The sequences of homologous genes from the archaeon Pyrococcus horikoshii were used to identify MPGS and MPGP genes in T. thermophilus HB27 genome. Both genes were separately cloned and overexpressed in Escherichia coli, yielding 3 to 4 mg of pure recombinant protein per liter of culture. The molecular masses were 43.6 and 28.1 kDa for MPGS and MPGP, respectively. The recombinant MPGS catalyzed the synthesis of alpha-mannosyl-3-phosphoglycerate (MPG) from GDP-mannose and D-3-phosphoglycerate, while the recombinant MPGP catalyzed the dephosphorylation of MPG to MG. The recombinant MPGS had optimal activity at 80 to 90 degrees C and a pH optimum near 7.0; MPGP had maximal activity between 90 and 95 degrees C and at pH 6.0. The activities of both enzymes were strictly dependent on divalent cations; Mn(2+) was most effective for MPGS, while Mn(2+), Co(2+), Mg(2+), and to a lesser extent Ni(2+) activated MPGP. The organization of MG biosynthetic genes in T. thermophilus HB27 is different from the P. horikoshii operon-like structure, since the genes involved in the conversion of fructose-6-phosphate to GDP-mannose are not found immediately downstream of the contiguous MPGS and MPGP genes. The biosynthesis of MG in the thermophilic bacterium T. thermophilus HB27, proceeding through a phosphorylated intermediate, is similar to the system found in hyperthermophilic archaea.
Empadinhas
Nuno
N
Departamento de Bioquímica, Universidade de Coimbra, 3004-517 Coimbra, Portugal.
Albuquerque
Luciana
L
Henne
Anke
A
Santos
Helena
H
da Costa
Milton S
MS
D000595:Amino Acid Sequence; D019641:DNA, Archaeal / Q000032:analysis; D004269:DNA, Bacterial / Q000032:analysis; D005988:Glyceric Acids; D006358:Hot Temperature; D008358:Mannose / Q000031:analogs & derivatives* / Q000096:biosynthesis*; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D019711:Pyrococcus / Q000201:enzymology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D019641:DNA, Archaeal; D004269:DNA, Bacterial; 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