Abstract
BackgroundEstE1 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.ResultsHere we report for the first time the 2.1-Å resolution crystal structure of EstE1. The three-dimensional structure of EstE1 exhibits a classic α/β 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 α/β hydrolases. EstE1 exists as a dimer that is formed by hydrophobic interactions and salt bridges. Circular dichroism spectroscopy and heat inactivation kinetic analysis of EstE1 mutants, which were generated by structure-based site-directed mutagenesis of amino acid residues participating in EstE1 dimerization, revealed that hydrophobic interactions through Val274 and Phe276 on the β8 strand of each monomer play a major role in the dimerization of EstE1. In contrast, the intermolecular salt bridges contribute less significantly to the dimerization and thermostability of EstE1.ConclusionOur results suggest that intermolecular hydrophobic interactions are essential for the hyperthermostability of EstE1. The molecular mechanism that allows EstE1 to endure high temperature will provide guideline for rational design of a thermostable esterase/lipase using the lipolytic enzymes showing structural similarity to EstE1.
Highlights
EstE1 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
We recently reported a new hormone-sensitive esterase/lipase (HSL) family [7] hyperthermostable esterase, EstE1, which was isolated by functional screening of a metagenomic DNA librariy constructed from a thermal environment sample [5]
It exhibits an esterase activity on short chain acyl derivatives of length C4–C6 at temperatures of 30 to 90°C, and is stable at temperatures exceeding 90°C [5,8] The amino acid sequence of EstE1 is significantly similar to other thermostable HSL-family esterases, including an esterase from hyperthermophilic archaeon Pyrobaculum calidifontis (64%) [9], the esterase AFEST from hyperthermophilic archaeon Archeoglobus fulgidus (57%) [10], and the esterase EST2 from thermophilic bacterium Alicyclobacillus acidocaldarius (51%) [8] Recently, the three-dimensional (3D) structure of EstE1 was predicted by homology modeling using the AFEST esterase as a reference [8]
Summary
EstE1 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. Their extraordinary thermodynamic stability allows these enzymes to function in organic solvents, and at elevated temperatures that approach or exceed 100°C [5,6]. We recently reported a new hormone-sensitive esterase/lipase (HSL) family [7] hyperthermostable esterase, EstE1, which was isolated by functional screening of a metagenomic DNA librariy constructed from a thermal environment sample [5]. Structure and sequence-based analyses for the thermostability determinants of EstE1 identified multiple intramolecular ion-pair networks and hydrophobic interactions critical for the thermostability of EstE1 [8]
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