A unique highly thermostable 2-phosphoglycerate forming glycerate kinase from the hyperthermophilic archaeon Pyrococcus horikoshii: gene cloning, expression and characterization.
A glycerate kinase (GK) gene (PH0495) from the hyperthermophilic archaeon Pyrococcus horikoshii, was cloned and expressed in Escherichia coli. The recombinant protein was purified to homogeneity by affinity chromatography and ion exchange chromatography. The enzyme was likely a homodimer based on SDS-PAGE (47 kDa) and gel filtration chromatography (100 kDa) analysis. A radioisotope-labeling examination method was initially used for the enzymatic activity detection, and the enzyme (GK(ph)) was found to catalyze the formation of 2-phosphoglycerate using D: -glycerate as the substrate. The enzyme exhibited unique phosphoryl donor specificity with maximal activity towards pyrophosphate. The temperature and pH optima of the enzyme were 45 degrees C and 7.0, respectively, and about half of the maximal activity remained at 100 degrees C. The enzyme was highly thermostable with almost no loss of activity at 90 degrees C for 12 h. Based on sequence alignment and structural comparison it was assigned to group I of the trichotomy of GKs.
Liu
Bo
B
State Key Laboratory of Microbial Technology, Shandong University, 250100, Jinan, Shandong, People's Republic of China.
Hong
Ye
Y
Wu
Lei
L
Li
Zhuo
Z
Ni
Jinfeng
J
Sheng
Duohong
D
Shen
Yulong
Y
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D019281:Dimerization; D011756:Diphosphates / Q000378:metabolism; D004795:Enzyme Stability; D005988:Glyceric Acids / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043902:Pyrococcus horikoshii / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D019843:Archaeal Proteins; D011756:Diphosphates; D005988:Glyceric Acids; D008670:Metals; D011994:Recombinant Proteins; C008885:2-phosphoglycerate; D017853:Phosphotransferases (Alcohol Group Acceptor); C020670:glycerate kinase