Thermal stability of glucokinases in Thermoanaerobacter tengcongensis.
In the genome of Thermoanaerobacter tengcongensis, three genes belonging to ROK (Repressor, ORF, and Kinase) family are annotated as glucokinases (GLKs). Using enzyme assays, the three GLKs were identified as ATP-dependent GLK (ATP-GLK), ADP-dependent GLK (ADP-GLK), and N-acetyl-glucosamine/mannosamine kinase (glu/man-NacK). The kinetic properties of the three GLKs such as K(m), V(max), optimal pH, and temperature were characterized, demonstrating that these enzymes performed the specific functions against varied substrates and under different temperatures. The abundance of ATP-GLK was attenuated when culture temperature was elevated and was almost undetectable at 80°C, whereas the ADP-GLK abundance was insensitive to temperature changes. Using degradation assays, ATP-GLK was found to have significantly faster degradation than ADP-GLK at 80°C. Co-immunoprecipitation results revealed that heat shock protein 60 (HSP60) could interact with ATP-GLK and ADP-GLK at 60 and 75°C, whereas at 80°C, the interaction was only effectively with ADP-GLK but not ATP-GLK. The functions of GLKs in T. tengcongensis are temperature dependent, likely regulated through interactions with HSP60.
Beijing Institute of Genomics, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing 100101, China ; Developmental Genetics Section, Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA.
Zhong
Z
Qian
Jingjing
J
Zhao
Xue
X
Bai
Wei
W
Tong
Zhen
Z
Chen
Hanfu
H
Wei
Quanhui
Q
Wang
Siqi
S
Liu
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D015153:Blotting, Western; D018834:Chaperonin 60 / Q000378:metabolism; D004795:Enzyme Stability; D005941:Glucokinase / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D059748:Proteolysis; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D046968:Thermoanaerobacter / Q000201:enzymology*
D018834:Chaperonin 60; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D005941:Glucokinase