Preliminary Crystallographic Analysis Of Thioredoxin-dependent Thiol Peroxidase(SF2523) And Enzyme Activity Assay Of Flavonoids 3',5' Hydroxylase(F3'5'H)

Shigella flexneri was the most common pathogen causing bacillary dysentery.Thioredoxin-dependent thiol peroxidase(SF2523)proteins was from Shigella flexneri 2a str.301.It belonged to thioredoxin peroxidase family and played an important role in protecting the biological macromolecule by scavenging active oxygen generated in the process of aerobic metabolism.To understand the underlying mechanism,prokaryotically expressed thioredoxin-dependent thiol peroxidase protein was purified using affinity chromatography and gel filtration,crystallized using the vapour-diffusion method.The crystal grew in a condition consisting of 1.8mol/L tri-Ammonium citrate,pH 7.0 using 1 g/L protein solution at 289 K.A complete data set was collected from a crystal to 2.75 A resolution using synchrotron radiation at 100 K.The crystal belonged to space group P212121,with unit-cell parameters a=35.80 A,b=50.63 A,c=88.52 A,?=????90.00°.One molecule was found in the asymmetric unit with a Matthews coefficient of 2.03 A3/u,corresponding to a solvent content of 39.56%.Flavonoids 3',5' hydroxylase(F3'5'H)was a key enzyme for the synthesis of delphinidin in anthocyanin biosynthetic pathway.In order to obtain pure soluble protein,enzyme activity assay and crystallographic experiment,the expression vector of F3'5'H gene was constructed and screened.Finally,pure protein solution was obtained and the activity in vitro was determined.Crystallization experiments were in progress.