Expression Purification And Crystal Structure Analysis Of Key Enzymes TchmY And TchmZ In The Synthetic Pathway Of Moenomycin

Moenomycins are phospholipid antibiotics with good toxicity selectivity and low drug resistance.These compounds hold great potential in further development of novel antibiotics.However,the poor pharmacokinetic parameters of the moenomycin hindered its development as a new type of antibiotic.Previous studies have shown that the terminal fatty chain in the structure of moenomycin has a crucial influence on the activity and water solubility of the moenomycin.Through the study of the fatty chain in the process of the synthesis of the moenomycin,a better understanding of its structure and function is of great significance for enriching the configuration of the moenomycin and screening for available antibiotics.This study is based on the prenylcyctase TchmY and the prenylsynthetase TchmZ derived from Actinoplanes teichomyceticus.These two enzymes are involved in the synthesis of the aromatin fatty chain.The genes encoding TchmY and TchmZ were obtained by chemical synthesis and were cloned into pET-46 Ek/LIC vector and pET-32 Xa/LIC,respectively,the plasmids were transformed into E.coli BL21(DE3).Target protein was purified by Ni-NTA affinity chromatography and gel filtration chromatography.The protein purity was determined by SDS-PAGE,and the crystal growth conditions were screened by the setting-drop method the vapor phase diffusion method.After the growth conditions of the protein,the growth conditions were optimized.The protein crystal structure is analyzed by X-ray crystal diffraction.The crystal structure of TchmY was obtained for the first time.X-ray diffraction data show that the TchmY protein is orthorhombic,the space group is P3121,and the resolution is 1.52 A,unit-cell a=73.47 A,b=73.47 A,c=124.23 A,a=90°,β=90°,γ=120°.The phase of TchmY is solved by soaking the mercury iodine compound,derived data were resolved using a single isomorphic replacement of heterosonic scattering(SIRAS).Initial model was generated by using PHENIX’s AutoBuild module,the structure were further built by manual reconstruction using COOT,and structural refinement using PHENIX.Based on a native form of 1.52 A resolution dataset,the final model gives Rwork and Rfree of 0.166 and 0.187.This study analyzed the crystal structure of the prenylcyctase TchmY derived from Actinomycetes teichomyceticus,and laid a theoretical foundation for the synthesis of terminal lipid chains in the biosynthesis of metnomycin,which has important biological significance.