A chimeric inorganic pyrophosphatase derived from Escherichia coli and Thermus thermophilus has an increased thermostability

Factors contributing to the thermostability of inorganic pyrophosphatase (P Pase) were investigated by examining chimeric PPases from Escherichia coli and Thermus thermophilus (Tth). Two chimeric PPase genes, T1-135E (residues 1-135 from the N terminus are comprised of Tth PPase and residues 136-173 are derived from the C terminus of E. coli PPase) and T1-149E [residues 1-1 49 from the N terminus are from Tth PPase and the rest (150-175) are from E . coli PPase], were constructed by random chimeragenesis. After the genes w ere overexpressed in the E. coli BL21(DE3) strain and the expression produc ts were purified, we compared the characteristics of these chimeric PPases with those of the parental PPases. We found that the two chimeras had highe r activity than either parent PPase at the optimum temperature. We also exa mined thermal stability in terms of CD spectra, fluorescence spectra, and t hermal changes in enzyme activity. The results revealed that the thermal st ability of T1-149E is similar to that of Tth PPase, but T1-135E is much mor e stable. This suggests that the four residues that are different between T 1-135E and T1-149E may be critical for thermostability between the two chim eras. By comparing the three-dimensional structures of Tth and E. coli PPas es, we deduced that the following two factors may contribute to differences in thermostability. (1) Two residues (Thr138 and Ala141 in the Tth PPase a nd His140 and Asp143 in the E. coli PPase) in the vicinity of the trimer-tr imer interface were different. (2) The Ala144-Lys145 loop in the Tth PPase was deleted in the E. coli PPase and also in the T1-135E chimera. Therefore , we conclude that T1-135E was thermostabilized by these two factors, and a lso, the Tth PPase moiety may contribute to the structural integrity of the chimeric enzymes.