Ion pairs involved in maintaining a thermostable structure of glutamate dehydrogenase from a hyperthermophilic archaeon.
Intersubunit ion pairs are considered to be involved for maintaining a stable structure of the glutamate dehydrogenase (GDH) from hyperthermophiles. In order to demonstrate an effect of intersubunit ion pairs on the structural stability, two kinds of mutation (T138E, Thr at position 138 was replaced by Glu; E158Q, Glu at position 158 was replaced by Gln) which add and remove ion pairs, respectively, were introduced into Pk-gdhA gene encoding GDH from Pyrococcus kodakaraensis KOD1. Addition of one ion pair (Pk-GDHA-T138E) increased the optimum temperature and thermostability. In contrast, Pk-GDH-E158Q showed lower optimum temperature and less thermostability than wild type GDH. Structure analysis of GDHs was performed by circular dichroism (CD) and indicated that all recombinant enzymes (Pk-GDH, Pk-GDH-T138E, Pk-GDH-E158Q) possess different structures from that of natural GDH. Upon heat treatment (60 degrees C, 2 h), the structures of Pk-GDH and Pk-GDH-T138E were converted to another form close to the natural structure. However, no structural conversion by heat treatment was observed in Pk-GDH-E158Q. These results indicate that intersubunit ion pairs play an important role in forming thermostable structure of Pk-GDH.
Rahman
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Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan.
Fujiwara
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S
Nakamura
H
H
Takagi
M
M
Imanaka
T
T
D000595:Amino Acid Sequence; D001120:Arginine; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli; D005969:Glutamate Dehydrogenase / Q000737:chemistry*; D006358:Hot Temperature; D007477:Ions; D008239:Lysine; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D019711:Pyrococcus / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry; D013816:Thermodynamics
D007477:Ions; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D001120:Arginine; D005969:Glutamate Dehydrogenase; D008239:Lysine