Discovery Of Steroid Carboxymethyltransferase And Chemical-enzymatic Synthesis Of Physoperuvine

Steroids are a class of compounds with important physiological functions.Steroid drugs have great clinical application value.Methyltransferase is a kind of transferase widely distributed in organisms,in which O-methyltransferase is the most abundant one.By methylating the oxygen atoms of compounds produced in organisms’ metabolism,O-methyltransferases can significantly improve their biology or physical activity and also chemical reactivity.At present,the discovery and application of Omethyltransferases are concentrated on hydroxyl O-methyltransferases,while the research on carboxyl O-methyltransferases is relatively rare.In the first part of the study,we obtained enzymes catalyzing carboxymethylation of steroid 4-BNC side chains from Mycobacterium neoaurum and M.smegmatis respectively by sequence alignment,gene cloning,protein expression and activity assay.Subsequently,we studied the substrate scope of these two proteins.The result showed that they possess catalytic activity for a series of small molecular aromatic carboxylic acids.Furthermore,we found a protein(ML2640c)with similar sequence in the protein structure database by sequence alignment.Activity measurements showed that this protein also have the carboxymethylation activity.Using molecular docking,we established the interaction model between ML2640 c and substrates.The protein mutation experiment confirmed the reliability of the model.This study reveals a possible pathway of sterol metabolism in Mycobacterium,and provides a new tool for structural modification of steroids,what’s more,adds new members to the carboxymethyltransferases family,and provides a useful reference for the modification of such methyltransferases.Tropinane alkaloids have analgesic,anesthetic and other biological activities,which are important clinical drugs.At present,the acquisition of tropane alkaloids is mainly depend on plant extraction,chemical synthesis and microbial synthesis have made some progress,but they are also subject to many restrictions.Tropane is the core framework of tropane alkaloids,and physoperuvine is a kind of simple tropane alkaloid with only one hydroxyl group beyond tropane.It is expected that tropane derivatives can be obtained from physoperuvine.Up to now,physoperuvine has been synthesized by chemical methods.Imine reductases are a class of oxidoreductases mainly dependent on NAD(P)H.In recent decade,they have shown great potential in the synthesis of chiral amines,thus many imine reductase libraries have been established.In the second part,five imine reductases catalyzing cycloheptanone to produce physoperuvine were screened from our laboratory’s imine reductase library,and some of them showed good stereoselectivity.By optimizing the methylamine equivalent ratio,these enzymes showed high conversion efficiency.In addition,we applied the recently reported new imine reductases(Asp Red Am and Ad Red Am)to this study.Asp Red Am exhibited very high catalytic activity and stereoselectivity.Finally,we mutated the key site of imine reductases to further enhance its stereoselectivity.This study provides a simple route for the synthesis of physoperuvine,provides a new way for the synthesis of tropane alkaloids,and has important reference value for the application and modification of imine reductase.