Analysis of thermal adaptation in the HSL enzyme family.

The recently solved 3D structures of two thermostable members of the carboxylesterase/lipase HSL family, namely the Alicyclobacillus (formerly Bacillus) acidocaldarius (1) and Archaeoglobus fulgidus carboxylesterases (2) (EST2 and AFEST respectively) were compared with that of the mesophilic homologous counterpart Brefeldine A esterase from Bacillus subtilis (BFAE) (3). Since the 3D homology models of other members of the HSL family were also available, we performed a structural alignment with all these sequences. The resulting alignment was used to assess the amino acid "traffic rule" in the HSL family. Quite surprisingly, the data were in very good agreement with those recently reported from two independent groups and based on the comparison of a huge number of homologous sequences from the genus Bacillus, Methanococcus and Deinococcus/Thermus. Taken as a whole, the data point to the statistical meaning of defined amino acid conversions going from psychrophilic to hyperthermophilic sequences. We identified and mapped several such changes onto the EST2 structure and observed that such mutations were localized mostly in loops regions or ?-helices and were mostly excluded from the active site. A site-directed mutagenesis of two of the identified residues confirmed they were involved in thermal stability (4). These residues were involved in a salt bridge and a network of ion pairs. Therefore we adopted an "alanine-scanning" mutagenic approach in order to measure the contribution of residues involved in ion pairs and network of ion pairs that were conserved in EST2 and AFEST but not in BFAE to the enzyme stability. Fifteen single and five double mutants were produced, the proteins expressed in E. coli purified and characterised kinetically and structurally. Most of mutations were demonstrated to affect enzyme stability as well as kinetic properties. Data will be discussed in the context of the known EST2 3D structure. Experiments of "double-mutant cycle" are currently ongoing in order to dissect the electrostatic from others, concurrent, effects.