Thermus thermophilus無機ピロリン酸加水分解酵素の三量体間界面改変による耐熱化

概要

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Inorganic pyrophosphatase (EC. 3.6.1.1) from Thermus thermophilus (Tth PPase)is a thermostablehomohexamer of 174 amino acids，and its intertrimer interface is formed mainly by the symmetric α-helix Abetween subunits. Amino acids and their interactions composing intertrimer interface are different in hexamericFamily I PPases，and then it was deduced that Tth PPase showed high thermostability because of stabilizing thisinterface by interactions of these residues. In this study，we focused on Thr138 and Ala141 residues in intertrimerinterface of Tth PPase to confirm the relationship between intertrimeric residues and thermostability， and thenimproved 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, andquaternary structure in the wild type Tth PPase and all variants. Especially，about 38% of hexamer and almost40% of enzyme activity were observed in HD variant after heating even at 85℃. Therefore，we suggested thatthe conversion to a set of ionic His138 and Asp141 at intertrimer interface had increased the thermostability ofTth PPase，and then suppressed its thermal aggregation.
徳島大学の論文