Heat effect on the structure and activity of the recombinant glutamate dehydrogenase from a hyperthermophilic archaeon Pyrococcus horikoshii.
Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290+/-8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90 degrees C, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100 degrees C was 4 h). The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. K(m) values of the recombinant enzyme are comparable for both substrates: 0.2 mM for L-glutamate and 0.53 mM for 2-oxoglutarate. The enzyme was activated by heating at 80 degrees C for 1 h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent.
Wang
Shiyu
S
Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, 130023, Changchun, People's Republic of China.
Feng
Yan
Y
Zhang
Zuoming
Z
Zheng
Baisong
B
Li
Na
N
Cao
Shugui
S
Matsui
Ikuo
I
Kosugi
Yoshitsugu
Y
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D021122:Protein Subunits / Q000737:chemistry; D019711:Pyrococcus / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013816:Thermodynamics
D017931:DNA Primers; D021122:Protein Subunits; D011994:Recombinant Proteins; D005969:Glutamate Dehydrogenase