Characterization and crystal structure of the thermophilic ROK hexokinase from Thermus thermophilus(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
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概要
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We characterized and determined the crystal structure of a putative glucokinase/hexokinase from Thermus thermophilus that belongs to the ROK (bacterial repressors, uncharacterized open reading frames, and sugar kinases) family. The protein possessed significant enzymatic activity against glucose and mannose, with V_<max> values of 260 and 68 μmol・min^<-1>mg・^<-1> protein, respectively. Therefore, we concluded that the enzyme is a hexokinase. However, the hexokinase showed little catalytic capacity for galactose and fructose. Circular dichroism measurements indicated that the enzyme was structurally stable at 90℃. The crystal structure of the enzyme was determined at a resolution of 2.02 Å, with R_<cryst> and R_<free> values of 18.1% and 22.6%, respectively. The polypeptide structure was divided into large and small domains. The ROK consensus sequences 1 and 2 were included in the large domain. The cysteine-rich consensus sequence 2 folded into a zinc finger, and the bound zinc was confirmed by both electron density and X-ray absorption fine structure (XAFS) spectrum. The overall structure was a homotetramer that consisted of a dimer of dimers. The accessible surface area buried by the association of the dimers into the tetrameric structures was significantly higher in the T. thermophilus enzyme than in a homologous tetrameric ROK sugar kinase.
著者
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Mine Shouhei
National Institute Of Advanced Industrial Science And Technology (aist)
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NAKAMURA Tsutomu
National Institute of Advanced Industrial Science and Technology (AIST)
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Uegaki Koichi
National Institute Of Advanced Industrial Science And Technology (aist)
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Kashima Yasuhiro
Thermostable Enzyme Laboratory Co. Ltd.
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Nakamura Tsutomu
National Inst. Advanced Industrial Sci. And Technol. Osaka Jpn
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Nakamura Tsutomu
産業技術総合研究所
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Uegaki Koichi
National Inst. Of Advanced Industrial Sci. And Technol.
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Oku Takashi
Thermostable Enzyme Laboratory Co. Ltd.
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- Characterization and crystal structure of the thermophilic ROK hexokinase from Thermus thermophilus(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
- Characterization and crystal structure of the thermophilic ROK hexokinase from Thermus thermophilus