Structrual And Functional Identification Of An Organic Hydroperoxide Resistance Protein From Mycoplasma Pneumoniae

Objective: This study was to identify whether recombinant protein rMPN668 degraded organic hydroperoxide, and analyze the structure of rMPN668 by molecular modeling and explore the correlation between its structure and function in order to further comprehend the pathogenesis of Mp.Methods:mpn668 gene sequence was obtained from Genbank database, and amplified by polymerase chain reaction (PCR) on the basis of Mp129 genome DNA as template and specific primers designed by PRIMER5.0, and subcloned into vector pGEX-6P-1 with tac promoter and transformed this recombinant plasmid into E. coli BL21, then obtained overexpressive fusion protein with GST tag when induced by isopropylβ-D-1-thiogalactopyranoside (IPTG) and cleaved GST tag to harvest the target protein rMPN668, then detected the concentration of rMPN668 by BCA method, and organic hydroperoxidase activity by ferrous oxidation xylenol orange(FOX) assay. Mp129 strains were grown in the SP4 medium to the logarithmic growth phase, then placed in the solid medium and cultured for 3-5d under oxidative stress, and extracted the total RNA of Mp129 strains, and detected expressive level of mpn668 mRNA by RT-PCR. Simulated the molecular model of rMPN668 by software Expasy and Visual Molecular Dynamics(VMD)through methods of homology modeling and molecular dynamics. Then performed site-directed mutagenesis of the amino acid in predicted protein active site, and harvested the mutant expression product by induction and purification, and detected organic hydroperoxidase activity by FOX assay.Results:423bp target gene was amplified successfully. The results of PCR, restriction enzyme digestion and DNA sequencing indicated the insereted fragment of prokaryotic recombinant plasmid was consistent with M129 mpn668 DNA logged in the Genbank. The soluble expressive product about 41KD was obtained when induced by isopropylβ-D-1-thiogalactopyranoside (IPTG). After purified by GST?Bind? Purification Kit, we harvested GST fusion protein and its purity can reach up to 95%. The GST tag was successfully cleaved by PreScission Protease. rMPN668 can degraded both inorganic and organic hydroperoxides, such as tert-butyl hydroperoxide(t-BHP)and H2O2. The degradation ratio of organic hydroperoxides and inorganic hydrogen peroxide was positively correlated with the concentration of rMPN668. when catalyzing t-BHP, 20ng/μL rMPN668 eliminated about 500μM t-BHP within 2min; however, the catalytic efficiency under interaction with H2O2 was much lower than this level, and 200ng/μL rMPN668 degraded 500μM H2O2 beyond 35min. RT-PCR analysis showed that expression of mpn668 in Mp in a certain range (t-BHP 0.05M~1.00M, CHP 0.05M~0.65M) increased with the increment of oxidant concentration. The modeling structure shows that secondary structural sequence of rMPN668 wasβ1-β2-β3-α1-β4-β5-α2-α3-β6, and Cys55 was likely located in protein active site and displayed organic hydroperoxidase activity. After Cys55 was missensibly mutated, the enzymatic activity of expressive product was almost lost, which initially confirmed that Cys55 was likely located in the active site.Conclusions:(1) rMPN668 displayed hydroperoxidase activity towards both inorganic and organic hydroperoxides, and degradation ratio of the later is higher than the former .(2) expression of mpn668 in M. pneumoniae was upregulated in response to oxidative stress.(3) Cys55 was likely located in protein active site and played the important role in the enzymatic reaction.