Monday, July 30, 2007 - 3:00 PM
S56
Crystal Structure and Biochemical Properties of a Thermostable Esterase
Mark Levisson1, Lei Sun2, Sjon Hendriks1, Twan Akveld1, Robert H.H. van den Heuvel3, Bauke W. Dijkstra2, John van der Oost1, and Servé W.M. Kengen1. (1) Laboratory of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, Wageningen, 6703 CT, Netherlands, (2) Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, Groningen, 9747 AG, Netherlands, (3) Biomolecular Mass Spectrometry, Utrecht University, Sorbonnelaan 16, Utrecht, 3584 CA, Netherlands
A bioinformatic screening of the genome of the hyperthermophilic bacterium Thermotoga maritima for ester-hydrolyzing enzymes revealed a hypothetical protein with typical esterase motifs. The predicted esterase (EstA) was cloned, functionally expressed in E. coli and purified to homogeneity. EstA is a thermostable esterase that showed optimal hydrolysis of esters with short acyl chains (C2-C8). The monomeric enzyme has a molecular mass of 43 kDa and optimal activity around 90°C and at pH 8.5. Analysis using mass-spectrometry and dynamic light scattering showed that EstA exists as hexamer. The crystal structure has been determined at 2.5 Å resolution using multiple anomalous dispersion data. The crystal structure showed a classical alpha/beta hydrolase domain containing the catalytic triad consisting of a serine, aspartate and histidine, and a new immunoglobulin-like domain. EstA was found to be inhibited by the organophosphate paraoxon. The structure of EstA with paraoxon bound was determined at 3.0 Å resolution and revealed that the inhibitor binds covalently to the catalytic serine residue. The structural information for EstA provides insight into the molecular basis of substrate recognition and catalysis by this enzyme, thereby contributing to the development of novel thermostable esterases to be used in industry.
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