Effect of Heat Treatment on Proper Oligomeric Structure Formation of Thermostable Glutamate Dehydrogenase from a Hyperthermophilic Archaeon
Shinsuke FUJIWARA*a, Raja N. Z. A. RAHMANc, Masahiro TAKAGIa, Shigenori KANAYAb, and Tadayuki IMANAKAc
a Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565, Japan
b Department of Material and Life Sciences,Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565, Japan
c Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 606-01, Japan
Large amounts of protein can be expressed as a recombinant protein by introducing a cloned gene in Escherichia coli. However, some recombinant proteins show the different properties from those of natural ones. The exact reasons of such differences have been unclear. Structural differences between the two may cause some distinction, however this idea has not been proved. We are interested in the structural and enzymatic differences between natural and recombinant enzymes from hyperthermophile. In order to gain better understanding, heat effect on the structure of recombinant enzyme was examined in comparison with that of natural one. In addition, enzyme characteristics of recombinant form with and without heat treatment were compared with those of natural form.
Natural glutamate dehydrogenase (Pk-GDH) was purified from hyperthermophilic archaeon Pyrococcus Sp. KOD1 to homogeneity and its activity and structure were compared with those of recombinant enzyme, which was expressed in E. coli. Determination of the molecular weight of these enzymes by SDS-PAGE and the gel filtration revealed that the natural enzyme was purified only as a hexameric form, whereas the recombinant enzyme was purified both as a monomeric and hexameric forms. Determination of the enzymatic activities indicated that only the enzyme in a hexameric form is active. Moreover, it is noted that the specific activity of the hexameric form of the recombinant enzyme is much lower than that of the natural enzyme and circular dichroism (CD) spectra of these enzymes are distinctly different from each other. These results suggest that the structure of the hexameric form of the recombinant enzyme with low specific activity (Type I) is different from that of the natural enzyme with high specific activity (Type II). Upon heat treatment (80℃, 15 min), the Type I structure was effectively converted to Type II structure and the specific activity of the enzyme was increased by 2.6 fold.Likewise, upon heat treatment (70℃ for 15 min), the inactive monomeric form of the recombinant enzyme was at least partially associated to hexameric form. These results indicate that high temperature plays an important role for proper folding and oligomerization of Pk-GDH.
