A Novel Candidate for the True Fructose-1,6-bisphosphatase in Archaea

• Published in: The Journal of biological chemistry 2002-08, Vol.277 (34), p.30649-30655
• Main Authors: Rashid, Naeem, Imanaka, Hiroyuki, Kanai, Tamotsu, Fukui, Toshiaki, Atomi, Haruyuki, Imanaka, Tadayuki
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Archaea - enzymology; Archaeal Proteins - metabolism; Chromatography, High Pressure Liquid; Cloning, Molecular; Fructose-Bisphosphatase - chemistry; Fructose-Bisphosphatase - genetics; Fructose-Bisphosphatase - metabolism; Hydrogen-Ion Concentration; Molecular Sequence Data; Molecular Weight; Temperature; Transcription, Genetic
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M202868200

Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes of the gluconeogenic pathway. Although enzyme activity had been detected in Archaea, the corresponding gene had not been identified until a presumable inositol monophosphatase gene from Methanococcus jannaschii was found to encode a protein with both inositol monophosphatase and FBPase activities. Here we display that a gene from the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1, which does not correspond to the inositol monophosphatase gene from M. jannaschii , displays high FBPase activity. The FBPase from strain KOD1 was partially purified, its N-terminal amino acid sequence was determined, and the gene ( Tk-fbp ) was cloned. Tk-fbp encoded a protein of 375 amino acid residues with a molecular mass of 41,658 Da. The recombinant Tk-Fbp was purified and characterized. Tk-Fbp catalyzed the conversion of fructose 1,6-bisphosphate to fructose 6-phosphate following Michaelis-Menten kinetics with a K m value of 100 μ m toward fructose 1,6-bisphosphate, and a k cat value of 17 s −1 subunit −1 at 95 °C. Unlike the inositol monophosphatase from M. jannaschii , Tk-Fbp displayed strict substrate specificity for fructose 1,6-bisphosphate. Activity was enhanced by Mg 2+ and dithioerythritol, and was slightly inhibited by fructose 2,6-bisphosphate. AMP did not inhibit the enzyme activity. We examined whether expression of Tk-fbp was regulated at the transcription level. High levels of Tk-fbp transcripts were detected in cells grown on pyruvate or amino acids, whereas no transcription was detected when starch was present in the medium. Orthologue genes corresponding to Tk-fbp with high similarity are present in all the complete genome sequences of thermophilic Archaea, including M. jannaschii , Pyrococcus furiosus , Sulfolobus solfataricus, and Archaeoglobus fulgidus , but are yet to be assigned any function. Taking into account the high FBPase activity of the protein, the strict substrate specificity, and its sugar-repressed gene expression, we propose that Tk-Fbp may represent the bona fide FBPase in Archaea.