Protein Thermostability Above 100℃: A Key Role for Ionic Interactions

　

C. VETRIANI^a, D. L. MAEDER^a, N. TOLLIDAY^a, K.-S. YIP^b, T.J. STILLMAN^b , K. L. BRITTON^b, D. RICE^b, H. H. KLUMP^c, and F. T. ROBB^a

　

^a Center of Marine Biotechnology, Columbus Center, 701 E Pratt Street, Baltimore, MD 21202, USA

^b Krebs Institute, University of Sheffield, England

^c Dept. of Biochemistry, University of Cape Town, Rondebosch 7700, South Africa

　

We have defined the minimal site-directed mutations necessary to elevate thermostability in the glutamate dehydrogenase (GIuDH) from the hyperthermophilic archaeon Thermococcus litoralis, an archaeon that grows optimally at 88℃. The mutation sites were deduced from consideration of the structure and amino acid sequence of the GluDH from Pyrococcus furiosus, a closely related archaeon growing optimally at 100℃.

An initial mutation (T138E) resulted in a GluDH With decreased thermostability. An additional complementary point mutation (D 167T) elevated the thermostability over the wild type enzyme. These experimental results provide strong support for a general mechanism of enzyme stabilization at very high temperature in which structural integrity is maintained by cooperative ionic interactions between subunits.

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