phosphofructokinase-B (MJ0406) from Methanocaldococcus jannaschii represents a nucleoside kinase with a broad substrate specificity

• Published in: Extremophiles : life under extreme conditions 2007-01, Vol.11 (1), p.105-114
• Main Authors: Hansen, Thomas, Arnfors, Linda, Ladenstein, Rudolf, Schönheit, Peter
• Format: Article
• Language: English
• Subjects: Aeropyrum pernix; Amino Acid Sequence; Archaea; Archaeal Proteins - chemistry; Archaeal Proteins - genetics; Archaeal Proteins - metabolism; Bacteria; Bacteriology; Biological and medical sciences; Cloning, Molecular; Desulfurococcus; DNA, Archaeal; E coli; Enzyme Stability; Enzymes; Escherichia coli; Fundamental and applied biological sciences. Psychology; Hyperthermophilic archaea; Kinetics; Metabolism. Enzymes; Methanocaldococcus jannaschii; Methanococcus - enzymology; Methanococcus - genetics; Microbiology; Molecular Sequence Data; Molecular Weight; Nucleoside kinase; Open Reading Frames; PFK-B; Phosphofructokinases - chemistry; Phosphofructokinases - genetics; Phosphofructokinases - metabolism; Phosphotransferases - chemistry; Phosphotransferases - genetics; Phosphotransferases - metabolism; Phylogeny; Protein Subunits; Recombinant Proteins - metabolism; Sequence Alignment; Substrate Specificity; Temperature; thermal stability
• ISSN: 1431-0651; 1433-4909
• DOI: 10.1007/s00792-006-0018-1

Recently, unusual non-regulated ATP-dependent 6-phosphofructokinases (PFK) that belong to the PFK-B family have been described for the hyperthermophilic archaea Desulfurococcus amylolyticus and Aeropyrum pernix. Putative homologues were found in genomes of several archaea including the hyperthermophilic archaeon Methanocaldococcus jannaschii. In this organism, open reading frame MJ0406 had been annotated as a PFK-B sugar kinase. The gene encoding MJ0406 was cloned and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 68 kDa composed of 34 kDa subunits. With a temperature optimum of 85°C and a melting temperature of 90°C, the M. jannaschii nucleotide kinase represents one of the most thermoactive and thermostable members of the PFK-B family described so far. The recombinant enzyme was characterized as a functional nucleoside kinase rather than a 6-PFK. Inosine, guanosine, and cytidine were the most effective phosphoryl acceptors. Besides, adenosine, thymidine, uridin and xanthosine were less efficient. Extremely low activity was found with fructose-6-phosphate. Further, the substrate specificity of closely related PFK-Bs from D. amylolyticus and A. pernix were reanalysed.