Enzymatic Mechanism Study In Phosphotransfer

Naturally produced molecules possessing a C–P bond–termed phosphonates or phosphinates–are a relatively rare and overlooked but remarkably successful class of natural products,most prominent representatives are the antibiotics fosfomycin and the herbicide glufosinate.However,the research on phosphonate natural products is still in its infancy.The reason is phosphonates lack chromophores and high polarity,which limits the use of HPLC for deep research.Even more overlooked are phosphonate modifications of cell surfaces,and biosynthetic knowledge of these tailoring pathways are extremely limited.To decipher these pathways,we are constructing phosphonate biosynthetic pathways.This thesis focus on phosphotransferase pathway（Pnt pathway）,which catalize biosynthesis and transfer of phosphonate related to cell surface modifications depend on bioinformatics analysis.Key enzymes of Pnt pathway from Olsenella uli and Treponema denticola:Oul-ppm,Oul-ppd,Tde-1415（Tde AEPT/Pnt C）,Oul-Pnt D were fully investigated.To decipher these pathways,we constructed phosphonate biosynthetic pathways for heterologous expression in BL21.Beside this Pnt pathway project,some side projects were investigated as well:1)NMR based C-P bond（phosphonate）rapid track method were installed;2)Kanamycin-induced production of 2’,3’-cyclic AMP in Escherichia coli.;3)Oral anaerobic bacteria inhibitory natural product discovery.Statement as follows:Prat 1:Pnt pathway key enzyme from O.uli and T.denticola enzymology1.Heterologous expression of O.uli genes Oul-ppm,Oul-ppd,isolation and purification of enzyme Oul-ppm,Oul-ppd,enzyme kinetics research and functional verification were performed.Oul-ppm catalize from Pnpy to PEP with enzyme kinetic k_cat=0.14±0.02 s^-1,Km=60±22μM;Oul-ppd catalize from Pnpy to Pn AA with enzyme kinetic k_cat=4.7±0.3 s^-1,Km=19±4μM.2.DFPP as inhibitor of Oul-ppd were organic synthesized.In vitro inhibit activity of DFPP to Oul-ppd was tested as IC₅₀=0.34μM.DFPP In vivo inhibit activity to Pnt pathway of O.uli wild-type was tested as well.However,no inhibitory activity were found to O.uli,which might because of DFPP was not able to get into live cell of O.uli.So we will keep on structure upgreading of DFPP to make sure it works with live cell.3.Bifunctional enzyme Tde1415（Tde-AEPT/Pnt C）of T.denticola were isolated and purified.Their catalize capacity were tested,by verification of product Pn AA and CMP-AEP.Product CMP-AEP was purified and saved as standard chemical.4.Multiple methods were tried for heterologous expression of Oul-Pnt D and enzyme Oul-Pnt D purification.However,as a membrane protein,Oul-Pnt D was expressed as inclusion body,which was not soluble all the time.Part 2:Multiple plasmid Pnt pathway heterologous expression system construction and study1.Phosphonate biosynthetic pathways for heterologous expression system BL21-Oul-ppm,Oul-ppd were constructed.Product was conformed as HEP by synthesis and adding HEP as internal standard.Biosynthesis mechanism of HEP in this system was proposed and conformed as well.2.Phosphonate biosynthetic pathways for heterologous expression system BL21-Oul-ppm,Oul-ppd,Tde1415-R15A were constructed.Product was conformed as AEP,which means all enzymes work well with each other in heterologous expression system.System BL21-Oul-ppm,Oul-ppd,Tde1415（Tde AEPT/Pnt C）were built as well,product were conformed as AEP and CMP-AEP.Multiple methods were used to rise CMP-AEP production and accumulation,together with CMP-AEP purification methord,an easy and cheap way to produce CMP-AEP was well installed.3.System BL21-Oul-ppm,Oul-ppd,Tde1415（Tde AEPT/Pnt C）,Oul-Pnt D was installed.In vivo catalize capacity of Oul-Pnt D was Investigated.Product was characterized by NMR and MASS,new natural product skeleton Ethoxy（hydroxymethyl）phosphinic acid（EHm P）was found as product,which indicates Oul-Pnt D participate catalize of CMP-AEP to EHm P.This Multiple plasmid heterologous expression system of Pnt pathway was well builted,which will be used in further Pnt pathway gene discovery.4.NMR based C-P bond（phosphonate）rapid track method were installed for project need.This methord will be a great gain for phosphonate researchers.Part 3:We isolated and elucidated the structure of 2’,3’-c AMP in E.coli,which appeared as a peak at 20.5 ppm by ³¹P NMR of cell extracts exposed to kanamycin.Although we were unable to detect 2’,3’-c AMP from extracts of cells grown in the presence of other antibiotics,its production cannot be ruled out due to the relative insensitivity of ³¹P NMR.Previous studies have shown separately that:aminoglycosides induce ribonuclease activity（Song et al.2014）,and ribonuclease activity leads to 2’,3’-c AMP production（Fontaine et al.2018b）.Here we present evidence directly linking the aminoglycoside kanamycin to 2’,3’-c AMP production.Part 4:Oral anaerobic bacteria O.uli inhibitory natural products were scaned,and C.globosum extraction was chosen as candidate.One new natural products chaetoglobol acid（1）together 11 known natural products were found in C.globosum extraction.However,none purified natural products showed O.uli inhibitory activity.Surprisingly,new natural product chaetoglobol acid showed good type II diabetes enzyme inhibitory activity,α-glucosidase IC₅₀=3.04μM andα-amylase IC₅₀=22.18μM.Above all,our work focused on phosphotransferase pathway.Key enzymes of Pnt pathway were fully investigated.Phosphonate biosynthetic pathways for heterologous expression in BL21 were built for the frist time and used in Pnt pathway investigation.As well as some side projects were investigated:NMR based C-P bond（phosphonate）rapid track method were installed for the frist time;Kanamycin-induced production of 2’,3’-cyclic AMP in Escherichia coli were investigated.;Last but not least,One new natural product process good type II diabetes enzyme inhibition activity was found from Chaetomium globosum.