Thermostable glycerol kinase from a hyperthermophilic archaeon: gene cloning and characterization of the recombinant enzyme

• Published in: Protein engineering 1998-12, Vol.11 (12), p.1219-1227
• Main Authors: Koga, Y, Morikawa, M, Haruki, M, Nakamura, H, Imanaka, T, Kanaya, S
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Sequence; Cloning, Molecular; Dimerization; Enzyme Stability; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Gene Expression; Glycerol Kinase - chemistry; Glycerol Kinase - genetics; Glycerol Kinase - metabolism; Hot Temperature; Humans; Hydrogen-Ion Concentration; Models, Molecular; Molecular Sequence Data; Molecular Weight; Pyrococcus - enzymology; Pyrococcus kodakaraensis; Recombinant Proteins; Sequence Alignment; Substrate Specificity
• ISSN: 0269-2139; 1741-0126; 1741-0134
• DOI: 10.1093/protein/11.12.1219

The Pk-glpK gene, which encodes glycerol kinase (GK) from a hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1, was cloned and expressed in Escherichia coli. The amino acid sequence of this enzyme (Pk-GK) deduced from the nucleotide sequence showed 57% identity with that of E. coli GK and 47% identity with that of human GK. Pk-GK, which has a molecular weight of 55902 (497 amino acid residues), was purified from E. coli and characterized. Despite the high sequence similarity, Pk-GK and E. coli GK are greatly divergent in structure and function from each other. Unlike E. coli GK, which exists as a tetramer, Pk-GK exists as a dimer. The preferred divalent cation for Pk-GK is Co2+, instead of Mg2+. The optimum pH and temperature for Pk-GK activity are 8.0 and 80 degrees C, respectively. Pk-GK can utilize other nucleoside triphosphates than ATP as a phosphoryl donor. It is fairly resistant to an allosteric inhibitor of E. coli GK, fructose-1,6-bisphosphate. Determination of the kinetic parameters indicates that the Km value of the enzyme is 15.4 microM for ATP and 111 microM for glycerol and its kcat value is 940 s(-1). The enzyme was shown to be fairly resistant to irreversible heat inactivation and still retained 50% of its enzymatic activity even after heating at 100 degrees C for 30 min. Construction of a model for the three-dimensional structure of the enzyme suggests that the formation of extensive ion-pair networks is responsible for the high stability of this enzyme.