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

摘要

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 37% identity with that of human GK, Pk-GK, which has a molecular weight of 55 902 (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 call 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 K-m value of the enzyme is 15.4 mu M for ATP and 111 mu M for glycerol and its k(cat) value is 930 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. [References: 52]