Structural And Functional Insight Into The FamC1 And FilC1 Cyclases From Stigonematales

Hapalindole-type indole alkaloids are a group of secondary metabolites from stigonematalean cyanobacteria which possess complex chiral carbon centers and show a broad spectrum of biological activities including antibacterial,anticancer and insecticidal.In recent years,the polycyclic system and its potential application value have attracted much attention and gradually become a research hotspot in the field of biochemical synthesis.In this dissertation,two members of Stigonematales cyclases(Stig cyclases),FamC1 and FilC1,were studied,which play an important catalytic role in the biosynthesis of Hapalindole indole alkaloids.Therefore,studies on the structure and function of these two enzymes are particularly important in the biosynthesis of Hapalindole alkaloids.In this dissertation,FamCl and FilC1 without transmembrane region were first expressed in E.coli BL21(DE3)host and then purified by two-step chromatographies of Ni-NTA and DEAE.Initial crystallization screening was performed by using Hampton Research Crystal Screens with the sitting-drop vapor diffusion method in the 48-well cryschem plate.After the preparation and collection of crystal diffraction datas of mercury derivatives,the initial electron density map of FamC1 was obtained by using single isomorphous replacement with anomalous scattering(SIRAS).The crystal structure of FamC1 was successfully solved by the refinements of Phenix and Coot.The crystal structure of FilC1 was solved by using molecular replacement with the structure of FamC1 as the template.In addition,in order to investigate the role of active site residues,the complex structures of FilCl/CI4 and FilCl/4IB were successfully obtained by soaking substrate analogues.Both FamCl and FilC1 crystal structures were ?-sandwich folds,which comprise antiparallel ?-strands arranged into two ?-sheets.Substrate-binding cavity is located in the variable loop regions.Besides,there are two calcium ions near the substrate-binding pocket.In addition,judging by the complex structures,the key catalytic residues were investigated and a possible mechanism of the formation of 12-epi-hapalindole U catalyzed by FamC1 or FilCl was proposed.In this dissertation,crystal X-ray diffraction technology was used to analyze the native and complex structures of FamC1 and FilC1 of Stig cyclases.According to the analysis of structure and mutagenesis,a possible mechanism of action of FamCl or FilC1 was proposed.These results provide important theoretical guidance to studies in other subfamilies of Stig cyclases and are of great significance in the biosynthesis of Hapalindole alkaloid.