Crystal Structural Analysis Of PaL And DehDIV-R

X-ray diffraction crystallography is the earliest technique used in the structure determination of biological macromolecules. It is now widely used in the enzyme structure analysis, which allows structural and functional study of the enzyme at the molecular level. Lipase and dehalogenase are important hydrolases. We plan to use X-ray diffraction crystallography solve their structures, and then study their catalytic mechanism and structure-function relationships.PaL belongs to bacterial lipase Family 1.3, obtained from Pseudomonas alcaligenes CGMCC4405 is a structurally and functionally novel lipase. It has the potential industrial application that catalyzes the mixture of 8 diastereomers of racemic menthol to yield L-menthol. We successfully expressed the PaL protein in Escherichia coli efficiently, and purified it by two-step column chromatography, obtained high-quality crystals by sitting-drop-vapor-diffusion method, and collected a set of reflection data at 1.8 A (at SSRF BL19U1). The structure was solved by single wavelengths anomalous scattering (SAD) and further refined by PHENIX and COOT. We found that the catalytic triplets of lipase are Ser164-His302-Asp274 by crystal structure analysis and Ser164 is in the GXSXG motif. PaL has two domains, and two specific loops which are more flexible than others. Enzyme assay shows that Loopl has steric hindrance effect and this assumption was validated by experiment. When two or four amino acids in Loop1 were cut off, the enzyme activity was improved.Dehalogenase DehDIV-R catalyzes the conversion of R,S-2-CPA to S-2-CPA. So far, no crystal structure of R-2-halogenated acid dehalogenase has been reported and the catalytic mechanism is unclear. DehDIV-R was expressed in Escherichia coli using pET-30a(+), pSJ-2, ppSUMO as vectors, and also purified it by column chromatography. High-quality crystals were obtained by sitting-drop-vapor-diffusion method, and collected a set of reflection data at 2.65 A (at SSRF BL18U1). We tried to solve the structure by molecular replacement (MR) and got the preliminary model.