ADP-dependent glucokinase/phosphofructokinase, a novel bifunctional enzyme from the hyperthermophilic archaeon Methanococcus jannaschii.
A gene encoding an ADP-dependent phosphofructokinase homologue has been identified in the hyperthermophilic archaeon Methanococcus jannaschii via genome sequencing. The gene encoded a protein of 462 amino acids with a molecular weight of 53,361. The deduced amino acid sequence of the gene showed 52 and 29% identities to the ADP-dependent phosphofructokinase and glucokinase from Pyrococcus furiosus, respectively. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized. To our surprise, the enzyme showed high ADP-dependent activities for both glucokinase and phosphofructokinase. A native molecular mass was estimated to be 55 kDa, and this indicates the enzyme is monomeric. The reaction rate for the phosphorylation of D-glucose was almost 3 times that for D-fructose 6-phosphate. The K(m) values for D-fructose 6-phosphate and D-glucose were calculated to be 0.010 and 1.6 mm, respectively. The K(m) values for ADP were 0.032 and 0.63 mm when D-glucose and D-fructose 6-phosphate were used as a phosphoryl group acceptor, respectively. The gene encoding the enzyme is proposed to be an ancestral gene of an ADP-dependent phosphofructokinase and glucokinase. A gene duplication event might lead to the two enzymatic activities.
Sakuraba
Haruhiko
H
Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Tokushima 770-8506, Japan.
Yoshioka
Issei
I
Koga
Shinji
S
Takahashi
Mamoru
M
Kitahama
Yuki
Y
Satomura
Takenori
T
Kawakami
Ryushi
R
Ohshima
Toshihisa
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D005941:Glucokinase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017017:Methanococcus / Q000201:enzymology*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D011994:Recombinant Proteins; C420842:ADP-dependent glucokinase; D017853:Phosphotransferases (Alcohol Group Acceptor); C442129:ADP D-fructose-6-phosphate 1-phosphotransferase; D005941:Glucokinase