Verification Of FAD Binding Site In GlpO And The Influence Of Gly On Activity Of GlpO From M.Ovipneumoniae

Mycoplasma ovipneumoniae (MO) is a major prokaryotic pathogen for sheep, which can lead to contagious pleuropneumonia.We found a glycerol metabolism genes glpO in Mycoplasma ovipneumoniae Y98 strain genome sequence (ID:73257),the gene full-length is 1,134 bp,encoding 377 amino acids.Glycerol-3-phosphate oxidase(GlpO) is a membrane protein,which can oxidized glycerol-3-phosphate to dihydroxy acetone phosphate(DHAP) and hydrogen peroxide(H2O2).The studies suggest that GlpO is an enzyme that belongs to the family of oxidoreductases which acting on the CH-OH group of the donor with oxygen as the acceptor.It employs one cofactor,flavin adenine dinucleotide (FAD).GlpO is one of pathogenic virulence factors of Mycoplasma pneumoniae, Mycoplasma mycoides subsp. mycoides SC PG1 Mycoplasma gallisepticum.It main involved in glycerol metabolic pathways, and oxidized glycerol-3-phosphate to H2O2,causing inflammation leading to cell death.Homologous alignment analysis found Y98 strains GlpO respectively 64%,62% similar with Mycoplasma mycoides subsp. mycoides SC PG1 GlpO and Mp GlpD.And there is also a possible FAD binding site structure in its 15-20 amino acid Gly15-Ala16-Gly17-Ile18-Ilei9-Gly2o.The research structure of GlpO FAD binding site suggest that Gly plays a key role to maintain the stability of the structure and its identification.Therefore,we obtained four kinds of FAD binding site amino acid deletion mutants by PCR amplification.For validating the FAD binding sites of MO GlpO,and to investigate different sites effect on its activity.The main results are as follows:① Using the pET28a-glpO vector as a template to obtain a FAD binding site deletion mutants glpOAFAD fragment through the large primer PCR method and ligated it to the pET32a expression vector,chose pET32a-glpO as a template by one-set opposite direction PCR method to get three kinds of FAD binding sites for a single Gly deletion mutant pET32a-glpO△Gly15, pET32a-glpO△Gly17, pET32a-glpO△Gly20.② Transformed four GlpO mutant recombinant plasmid into BL21 (DE3) host strain, induce the expression of recombinant proteins, detecting target protein about 60 kD are present in the form of inclusion bodies by SDS-PAGE and Western blot.The inclusion body proteins were dissolved in 6 M urea,successfully refolded mutant recombinant protein through dialyzing in concentration gradient of 4 M,2 M,0 M urea.③ Choose refolded protein GlpO as control to detect oxidation activity mutant proteins.Founding that GlpO protein having oxidase activity,it is possible to glycerol 3-phosphate as substrate catalytic produce H2O2 in the present of FAD,while no H2O2 produced in the absent of FAD.The four kinds GlpO FAD binding site deletion mutants recombinant protein without oxidase activitycan which can not catalyze glycerol 3-phosphate to H2O2 whether addition FAD or not.④ TC-1 cells was infected with MO, GlpO and GlpO mutant proteins to MTT and ROS assay,founding that whether adding glycerol or not,complete FAD binding site deletion mutants and FAD binding sites Gly single deletion mutants of recombinant protein had no effect on cell viability and will not cause the cells to produce ROS.In this study, by constructing four kinds of GlpO deletion mutants.Detecte recombinant protein activity at the protein and the cellular level,to verify the FAD binding site motify of GlpO,and proved that Gly15,Gly17,Gly20 are key sites for GlpO binding FAD.The results enrich the mycoplasma virulence factors relevant information,and provide basis for studing the pathogenesis of MO.