How structural features of MsrBs shed lights to the catalytic mechanism

Abstract

24 European Crystallographic Meeting, ECM24, Marrakech, 2007 Page s135 Acta Cryst. (2007). A63, s135 heart failure. The EST gene was cloned and expressed in E. coli; the recombinant protein is a non-disulfide linked homotrimer with a monomer molecular weight of 33 kDa in both solution and crystalline state, indicating that these ESTs function as trimers. EST hydrolyzed DL-MATI to produce DAT with the degree of conversion of 49.5 % and an enantiometric excess value 97.2 % at optimum pH 8~10 and temperature 57~67 C. The crystal structure of EST has been determined by X-ray diffraction to a resolution 1.6 A, confirming that EST is a member of the α/β hydrolase fold superfamily of enzymes and includes a catalytic triad Ser97, Asp227 and His256. The active site is located approximately in the middle of the molecule at the end of a pocket ~12 A deep. The EST-DL-MATI complex structure has also been determined and shows that the oxyanion hole can form by peptide NH groups of Thr98 and Trp31 that form hydrogen bonds with the carbonyl oxygen of the DL-MATI. The EST can hydrolyze methyl ester group without affecting the acetylthiol ester moiety in DL-MATI. The examination of substrate specificity of EST toward other linear esters revealed that the enzyme showed specific activity toward methyl esters and that it recognized the configuration at the C-2 position. The knowledge of substrate specificity, molecular recognition and structure of a substrate-binding site of EST is useful for enzymatic engineering for broader ester substrates.