Structural Studies On The Multifunctional Sam-dependent Lepi And Acetylhydroxyacid Synthase From The Hyperthermophilic Bacterium Thermotoga Maritima

Enzymes are indispensable and important substances for living organisms.They provide important guarantees for various catalytic reactions in organisms.With in-depth exploration,the application of biological enzymes in production and life is becoming more and more important.Pericyclic reactions are widely used in industrial synthesis,but few enzymes can catalyze the pericyclic reactions.At present,it has been reported that there are enzymes with similar activities in Aspergillus flavus.Exploring them will provide important value for industrial production.The typical organisms in extreme environments is Thermotoga maritime,which is more and more widely used in synthetic biology,and the research of its acetohydroxy acid synthase will provide a new direction for the production of branched-chain amino acids.In this study,we performed the structural studies into two important enzymes.One is the multifunctional SAM-dependent enzyme LepI,which is a key enzyme for generating pyran rings in the biosynthesis of leporinB pathway in Aspergillus flavus.By constructing recombinant plasmids and performing purification to obtain proteins with better purity and condition,crystallization experiments were conducted.After screening 768 conditions,crystals were found,and the relevant crystallization conditions were optimized.Valid diffraction data were obtained from X-Ray diffraction approaches.In the absence of homologous structures,through the purification and crystallization experiment of the SeMet protein,we successfully solved the structure of LepI.Combinded with structural analysis,sequence alignment and molecular docking,we suggest the working mechanism of LepI.The other enzyme in this study is the acetohydroxy acid synthase from Thermotoga maritima.Acetohydroxy acid synthase is the first rate-limiting enzyme in the process of biosynthesizing branched-chain amino acids.Generally,acetohydroxy acid synthase consists of two subunits,the large subunit,also called the catalytic subunit and the small subunit,also called the regulatory subunit.In this study,the recombinant plasmid was constructed for the catalytic subunit part,and the protein with different tags was successfully expressed.According to the relevant purification results,the selected His-tag proteins were subjected to crystallization experiments,and a total of 2544 conditions in the laboratory were screened.After the initial screen,some crystallization conditions were found,two better crystallization conditions were optimized and now the crystals diffracted with a 3.8 angstrom resolution.Subsequent optimization is still in progress.In summary,we solved the structure of LepI and proposed the structural biological basis of its detailed working mechanism,which provides the possibility for its future industrial applications.The exploration of the crystallization conditions of acetohydroxy acid synthase provides an important reference for its future crystal structure analysis.