A snapshot of a transition state analogue of a novel thermophilic esterasebelonging to the subfamily of mammalian hormone-sensitive lipase

EST2 is a novel thermophilic carboxylesterase, isolated and cloned from Ali cyclobacillus (formerly Bacillus) acidocaldarius, which optimally hydrolyse s esters with acyl chain lengths of six to eight carbon atoms at 70 degrees C. On the basis of the amino acid sequence homology, it has been classified as a member of the mammalian hormone-sensitive lipase (HSL) subfamily. The crystal structure of EST2, complexed with a sulphonyl derivative, has b een determined at 2.6 Angstrom resolution by a multiple wavelength anomalou s diffraction experiment on a seleno-methionine derivative. EST2 presents a canonical alpha/beta hydrolase core, shielded at the C-terminal side by a cap region built up of five helices. It contains the lipase-like catalytic triad, Ser155, His282 and Asp252, whereby the nucleophile is covalently mod ified. This allows an unambiguous view of the putative active site of EST2, detecting the oxyanion hole, in whose formation the amino acid sequence mo tif His81-Gly82-Gly83-Gly84 is involved, and the hydrophobic binding pocket for the acyl chain. The structural model here reported provides the first example of a transition state analogue of an esterase/lipase belonging to t he HSL group, thus affording useful information for the design of medical i nhibitors. Moreover, as the first X-ray structure of a thermophilic carboxy lesterase, the comparison with its mesophilic homologue, the Brefeldin A es terase (BFAE) from Bacillus subtilis, allows the identification of putative determinants of thermal stability. (C) 2000 Academic Press.