Structural Determination Of Flavin-dependent Monooxygenase AsFMO1 And Non-heme Monoferrase OvoA

Natural products are important sources for new drug development.Studies on natural product biosynthesis are to reveal the synthetic pathway at the gene and protein level,and to illustrate the molecular mechanism of biochemical process and enzymatic catalysis.This thesis work includes the structure determination of two enzymes,a flavin-dependent monooxygenase AsFMO1 and a non-heme monoferrase Ovo A.The first part of my work is the structure determination a flavin-dependent monooxygenase AsFMO1.In nature,alliin is a unique amino acid in garlic.It has unique pharmacological activities,including anti-tumor,lowering blood pressure,antibacterial and anti-virus,scavenging free radicals,protecting the liver,and anti-diabetics.Due to the extremely difficult extraction and preservation of alliin,currently only a few countries such as the United States,Switzerland and Canada can extract alliin efficiently.In 2015,Japanese biochemist Kazuki Saito successfully identified the biosynthetic gene cluster of alliin and proposed a complete biosynthetic pathway.AsFMO1 is a flavin-dependent monooxygenase,responsible for catalyzing the sulfur oxidation reaction in the biosynthesis of alliin.To deeply understand the biosynthesis mechanism of alliin,we did the structural work in collaboration of Professor Daniel Scharf in Zhejiang University.I successfully obtained high-purity recombinant AsFMO1 and the diffraction quality crystals.Two high resolution structures of AsFMO1 were solved with the molecular replacement method,the apo-AsFMO1 structure and the complex structure of holo-AsFMO1 with FAD,NADP~+and alliin.The second part of my work is to crystallize a non-heme monoferrase Ovo A.In1979,B.M.Shapiro isolated the natural antioxidant Ovothiol A from the extracellular fluid of the purple sea urchin Paracentrotus lividus,which was subsequently named Ovothiol A-C according to the number of methyl groups on the side chain of Ovothiol.We did the crystallization work of Methyloversatilis thermotolerans Ovo A in collaboration of associate professor Pinghua Liu at Boston University.I successfully obtained high-purity Ovo A-1-708,Ovo A-1-475 and Ovo A-466-708 crystallization experiments.The best crystal of Ovo A-466-708 and Ovo B product?-thiohistidine complex diffracted to 1.9?.However,I failed to determine the crystal structure due to low completeness of the dataset.