The phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus is a unique glycolytic enzyme that belongs to the cupin superfamily.
Pyrococcus furiosus uses a variant of the Embden-Meyerhof pathway during growth on sugars. All but one of the genes that encode the glycolytic enzymes of P. furiosus have previously been identified, either by homology searching of its genome or by reversed genetics. We here report the isolation of the missing link of the pyrococcal glycolysis, the phosphoglucose isomerase (PGI), which was purified to homogeneity from P. furiosus and biochemically characterized. The P. furiosus PGI, a dimer of identical 23.5-kDa subunits, catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate, with K(m) values of 1.99 and 0.63 mm, respectively. An optimum pH of 7.0 has been determined in both directions, and at its optimum temperature of 90 degrees C the enzyme has a half-life of 2.4 h. The N-terminal sequence was used for the identification of the pgiA gene in the P. furiosus genome. The pgiA transcription start site has been determined, and a monocistronic messenger was detected in P. furiosus during growth on maltose and pyruvate. The pgiA gene was functionally expressed in Escherichia coli BL21(DE3). The deduced amino acid sequence of this first archaeal PGI revealed that it is not related to its bacterial and eukaryal counterparts. In contrast, this archaeal PGI shares similarity with the cupin superfamily that consists of a variety of proteins that are generally involved in sugar metabolism in both prokaryotes and eukaryotes. As for the P. furiosus PGI, distinct phylogenetic origins have previously been reported for other enzymes from the pyrococcal glycolytic pathway. Apparently, convergent evolution by recruitment of several unique enzymes has resulted in the unique Pyrococcus glycolysis.
Verhees
C H
CH
Laboratory of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands. corne.verhees@algemeen.micr.wau.nl
Huynen
M A
MA
Ward
D E
DE
Schiltz
E
E
de Vos
W M
WM
van der Oost
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019766:Genes, Archaeal; D005956:Glucose-6-Phosphate Isomerase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006019:Glycolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D016366:Open Reading Frames; D020027:Pyrococcus furiosus / Q000201:enzymology*; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D005956:Glucose-6-Phosphate Isomerase