Novel type of glucose-6-phosphate isomerase in the hyperthermophilic archaeon Pyrococcus furiosus.
Glucose-6-phosphate isomerase (phosphoglucose isomerase [PGI]) (EC 5.3.1.9) from the hyperthermophilic archaeon Pyrococcus furiosus was purified 500-fold to homogeneity. The enzyme had an apparent molecular mass of 43 kDa and was composed of a single type of subunit of 23 kDa indicating a homodimeric (alpha(2)) structure. Kinetic constants of the enzyme were determined at the optimal pH 7 and at 80 degrees C. Rate dependence on both substrates followed Michaelis-Menten kinetics. The apparent K(m) values for glucose-6-phosphate and fructose-6-phosphate were 8.7 and 1.0 mM, respectively, and the corresponding apparent V(max) values were 800 and 130 U/mg. The enzyme had a temperature optimum of 96 degrees C and showed a significant thermostability up to 100 degrees C, which is in accordance with its physiological function under hyperthermophilic conditions. Based on the N-terminal amino acid sequence of the subunit, a single open reading frame (ORF; Pf_209264) was identified in the genome of P. furiosus. The ORF was characterized by functional overexpression in Escherichia coli as a gene, pgi, encoding glucose-6-phosphate isomerase. The recombinant PGI was purified and showed molecular and kinetic properties almost identical to those of the native PGI purified from P. furiosus. The deduced amino acid sequence of P. furiosus PGI did not reveal significant similarity to the conserved PGI superfamily of eubacteria and eucarya. This is the first description of an archaeal PGI, which represents a novel type of PGI.
Hansen
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T
Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 1-9, D-24118 Kiel, Germany.
Oehlmann
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M
Schönheit
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D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005956:Glucose-6-Phosphate Isomerase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000254:growth & development; D011817:Rabbits; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013696:Temperature
D005956:Glucose-6-Phosphate Isomerase