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An ionic interaction that holds an α-helix and a β-strand on which catalytic Asp and His residues are located, respectively, is conserved in a hyperthermophilic esterase EstE1 (optimum temperature 70 °C) and a mesophilic esterase rPPE (optimum temperature 50 °C). We investigated the role of an ionic interaction between E258 and R275 in EstE1 and that between E263 and R280 in rPPE in active-site stability of serine esterases adapted to different temperatures. Ala substitutions caused a 5–10 °C decrease in the optimum temperature of both EstE1 and rPPE mutants. Surprisingly, disruption of the ionic interaction caused larger effects on the conformational flexibility of EstE1 mutants despite their rigid structures, whereas the disruption had fewer effects on the thermal stability of EstE1 mutants at 60–70 °C, as the structure of EstE1 was adapted to high temperatures. In contrast, mesophilic rPPE mutants showed dramatic decreases in thermal stability at 40–50 °C, but less changes in conformational flexibility because of their inherently flexible structures. The results of this study suggest that the ionic interaction between the α-helix with catalytic Asp and the β-strand with catalytic His plays an important role in the active-site conformation of EstE1 and rPPE, with larger effects on the conformational flexibility of hyperthermophilic EstE1 and the thermal stability of mesophilic rPPE. |
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