Study On The Structure And Function Of R-2-Haloacid Dehydrogenase DehDIV-R And Lipase PaL

X-ray diffraction crystallography is the mainstream technology for analyzing the structures of biological macromolecules.It is widely used in analyzing three-dimensional structure of diverse enzymes.The interaction between enzymes and small molecules can be obtained by analyzing the crystal structures of enzymes and complexes.Combined with molecular dynamics simulation,the relationship between structures and functions of the enzyme can be studied from the molecular level,which provides a theoretical basis for the rational design of the enzyme.DehDIV-R is one kind of R-2-haloacid dehydrogenase from Pseudomonas ZJU6,which can hydrolyze R-2-chlorpropionic acid specifically.At present,there are few reports on the crystal structure of R-2-haloacid dehydrogenase,and the study of its substrate selectivity merely has an indirect explanation according to the structure of R,S-2-haloacid dehydrogenase.We purified DehDIV-R by multi-step chromatography successfully,obtained high-quality crystals by hanging drop vapour diffusion method,under the condition of 0.1 mol/L HEPES(pH 7.0),12%PEG 6000,0.2 mol/L MgCl2,8 mmol/L CHAPS,and collected a set of reflection data at 2.35 A in SSRF BU18U1.The crystal structure was solved by molecular replacement(MR)on CCP4 online server.The structural analysis shows that DehDIV-R is a pseudo-dimer consisting of multiple a helices,and the active center Asp205 is linked with Asnl 31 through hydrogen bonds,which is beneficial to the activation of water.Then,the molecular docking structure of DehDIV-R with R-or S-2-chlorpropionic acid was analyzed by restrictive molecular dynamics simulation.We speculated that the differences lead to the substrate selectivity of DehDIV-R in which exist in distances and angles between the oxygen atom of activated water and the C? of R-or S-2-chlorpropionic acid.PaL is one kind of lipase from Pseudomonas aeruginosa CGMCC4405,which can catalyze d,1-menthol propionate to produce optically pure 1-menthol,but it has low diastereoselectivity when it is used to catalyze four pairs of racemic menthol propionates.The crystal structure of PaL was obtained in the early experiments,but its catalytic traid is in an unreasonable position-His302 is located on the surface of enzyme.We tried to use various ways to get the structure of PaL with a reasonable catalytic traid.Finally,according to corse-grained molecular dynamics simulation,His302 was found to move in and out of the active pocket in many times.And then,the reasonable structure of PaL was obtained by the conventional molecular dynamics simulation.Based on this structure,molecular docking and molecular dynamics simulation were carried out to explore the reasons for the low diastereoselectivity of PaL.The chirality of four pairs of racemic menthol propionate at C1 affects the formation of hydrogen bonds between the protonated hydrogen atom of His302 and the oxygen atom of the substrate's hydroxyl,which determines whether it can be hydrolyzed.PaL can only identify the chirality at C1,not at C2 or C5,resulting in the poor diastereoselectivity of PaL.Therefore,it can not specifically hydrolyze 1-menthol propionate.