Thermostabilization by the Improvement of Intertrimeric Residues in Thermus thermophilus Inorganic Pyrophosphatase.

Inorganic pyrophosphatase (EC. 3.6.1.1) from Thermus thermophilus (Tth PPase)is a thermostable
homohexamer of 174 amino acids，and its intertrimer interface is formed mainly by the symmetric α-helix A
between subunits. Amino acids and their interactions composing intertrimer interface are different in hexameric
Family I PPases，and then it was deduced that Tth PPase showed high thermostability because of stabilizing this
interface by interactions of these residues. In this study，we focused on Thr138 and Ala141 residues in intertrimer
interface of Tth PPase to confirm the relationship between intertrimeric residues and thermostability， and then
improved their combination to His and Asp/Glu (HD or HE variant).
As results，the HD variant showed the highest thermostability of enzyme activity，fluorescence spectra, and
quaternary structure in the wild type Tth PPase and all variants. Especially，about 38% of hexamer and almost
40% of enzyme activity were observed in HD variant after heating even at 85℃. Therefore，we suggested that
the conversion to a set of ionic His138 and Asp141 at intertrimer interface had increased the thermostability of
Tth PPase，and then suppressed its thermal aggregation.