Structure And Function Of Thermophilic Esterase EstL5 In GDSL Family

Esterase is a type of enzyme that causes esters to split into an acid and an alcohol or synthesize an acid and an alcohol to generate ester-containing compounds. They play important roles both in life process and industrial applications.In this study, a novel moderately thermostable esterase, EstL5, was cloned from Geobacillus thermodenitrificans T2. It is identified as a new member of GDSL family esterase, and can be further classified into SGNH sub-family by sequence analysis. Construct containing full length sequence of EstL5cDNA was induced to be over-expressed in E. coli., and6His-tagged EstL5protein was purified. Enzyme activity analysis of EstL5showed preference of the enzyme for short-chain esters, especially p-nitrophenyl butyrate. We also detected relatively low activity of recombinant EstL5when hydrolyzes triglyeride esters, but no activity was detected with long-chain substrates. Therefore, we characterized EstL5as an esterase, not a lipase. Otherwise, recombinant EstL5hasn’t performed any activities as a thioesterase or a phosphatidase. The most suitable temperature for EstL5is60℃and the most suitable PH is8.0. Data also showed that EstL5retained24%activity when the reaction performed at0℃, and100%activity when incubated at55℃for12hours. EstL5can resist many kinds of organic solvent and compound. These physic-chemical property shows that EstL5possessed good stability and broad reaction temperature range, which provided it good prospect of application.To study the sequence and the structure of EstL5, purified SeMet-EstL5protein was crystallized by using the hanging-drop vapor-diffusion method and the data of X-ray diffraction were collected to a resolution of3.0A. The crystals of Se-EstL5belongs to the Primitive Tetragonal space group P43212, with unit-cell parameters a=b=101.48, c=124.56A. The crystal structure of SeMet-EstL5was solved by single-wavelength anomalous scattering. X-ray diffraction data of native EstL5were collected to a resolution of2.79A and the crystal structure was solved by using SeMet-EstL5as a model. The data showed little structural difference between EstL5and SeMet-EstL5. The structure data showed five parallel β-sheets located at the center of EstL5, both sides of which were covered by several α-helices. The structural characteristics of EstL5share same features with those of SGNH family esterases.The nucleophilic Ser63residue situates at the junction of a β-sheet and an α- helix, and the two remaining members of the catalytic triad, Asp235and His238, are in a loop region connecting two helices near the C terminus. They are very close with each other in the spatial structure. Ser63, Gly101and Asn133, which were predicted as oxyanion receptor, are part from each other with the distance from3.5to6A and perform as a loose oxyanion hole. Single mutation of these sites can greatly decrease the EstL5activity. We speculate that Ser-Asp-His catalytic triad is essential for the enzyme’s function, and the catalytic mechanism can be explained almost by oxyanion hole system.The structural and functional researches of EstL5were performed in this paper. EstL5was first cloned, purified and reported for its basic enzymal and physical feature. We successfully solved the crystal structure of EstL5and analyzed its possible mechanism. This would be the foundation for EstL5’s future application. And it would be helpful for those future researches of GDSL family esterase.