Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties

Background: EstEI is a hyperthermophilic esterase belonging to the hormone-sensitive lipase family and was originally isolated by functional screening of a metagenomic library constructed from a thermal environmental sample. Dimers and oligomers may have been evolutionally selected in thermophiles because intersubunit interactions can confer thermostability on the proteins. The molecular mechanisms of thermostabilization of this extremely thermostable esterase are not well understood due to the lack of structural information. Results: Here we report for the first time the 2.1-angstrom resolution crystal structure of EstEI. The three-dimensional structure of EstEI exhibits a classic alpha/beta hydrolase fold with a central parallel-stranded beta sheet surrounded by alpha helices on both sides. The residues Ser154, Asp251, and His281 form the catalytic triad motif commonly found in other alpha/beta hydrolases. EstEI exists as a dimer that is formed by hydrophobic interactions and salt bridges. Circular dichroism spectroscopy and heat inactivation kinetic analysis of EstEI mutants, which were generated by structure-based site-directed mutagenesis of amino acid residues participating in EstEI dimerization, revealed that hydrophobic interactions through Val274 and Phe276 on the beta 8 strand of each monomer play a major role in the dimerization of EstEI. In contrast, the intermolecular salt bridges contribute less significantly to the dimerization and thermostability of EstEI. Conclusion: Our results suggest that intermolecular hydrophobic interactions are essential for the hyperthermostability of EstEI. The molecular mechanism that allows EstEI to endure high temperature will provide guideline for rational design of a thermostable esterase/lipase using the lipolytic enzymes showing structural similarity to EstEI.