The Analysis Of The Structure And The Active Sites Of KsdD In Mycobacterium Neoaurum

3-ketosteroid-?1-dehydrogenase(KsdD)plays an indispensible role in the microbial catabolism of phytosterol(PS)to obtain the precursor of pharmaceutical steroid.Previously,the study about the active sites and the structure of KsdD mostly focus on the KsdD from Rhodococcus.However,when it comes to Mycobacterium which was the most widely used to obtain precursor from the side chain cleavage of PS,the structural information on KsdD is not yet available.In this study,we attempted to obtain the structural information on KsdD from Mycobacterium neoaurum TCCC 11028(MNR)by the way of purification,crystallization and X-ray crystallographic analysis and the way of site-directed mutagenesis under the direction of in-silico protein docking modeling to obtain information of the active sites and structure of KsdD-MNR.This study may serve as a basis for future studies on the structural analysis and catalytic mechanism of dehydrogenase.E.coli BL21(DE3)-pET-22b(+)-KsdD-MNR,E.coli BL21(DE3)-pET-15b-KsdD-MNR,E.coli BL21(DE3)-pET-28a-KsdD-MNR were constructed.SDS-PAGE using whole bacterial protein showed that KsdD-MNR was successfully expressed.Ni-NTA affinity chromatography,G-200 gel chromatographywy and as its like were used for the purification of KsdD.The result of BandScan5.0 showed the purity of KsdD is almost 70%.Sitting drop method was used for the crystallization of KsdD.But during crystallization process,we found it is hard to obtain the crystal which could meet the requirements of the preliminary X-ray crystallographic analysis of KsdD.3D structure of KsdD from MNR was homologically modeled using the MODeller 9.15 program(http://salilab.org/modeller/).The 3D structural information of AD,which was used as the ligand,was handled using Sybyl2.0 program.The compound AD was docked into the active site by the Syby12.0 program.The result suggested that Y122,Y125,S138,E140,Y365,Y541,Y472 in the substrate binding site of AD may play a key role in this transformation.Mutagenesis were constructed by OverlapPCR respectively.The conversion rate of AD by wild type and each mutations showed that the activity of KsdD was almost abolished in Y125,Y365 and Y541.While,Y122F,S138A and E140V contributed differently to the activity and Y472F did not affect the active of the enzyme.Meanwhile,swiss-modeller(http://swissmodel.expasy.org/)was used to build the site mutants' simulation of the conformation.Protein docking studies of AD to the modeled mutant enzyme structures combined the mechanism revealed that Y365,which was assisted by Y125 as general base to remove the hydrogen atom,and this maybe the indirect mechanism of Y122.Meanwhile,Y541 and G545 acted as electrophile to stablize the intermediate during this process.E138A removed the H bonds with Val-57,Val-139 and Pro-544,which was recognized to fix the movable loops.Not like the premeditation that E140 may have the direct reaction with AD,E140 may occupy a solvent-accessible pocket near the active site entrance collaborated with five-membered D-ring of the 3-ketosteroid.