Study On The Structure,Interactions And Enzyme Activity Of The SQR And Mutants From Thiobacillus Denitrificans ATCC25259

There are two ways which produce hydrogen sulfide.One is produced by nature and the other is produced by human activities.Hydrogen sulfide released by nature is less than 10% of global emissions.Therefore,most of the hydrogen sulfide emissions are caused by human production activities and daily life activities.There are a wide variety of strain sources of Thiobacillus denitrificans.Thiobacillus denitrificans grows under mild conditions and has the highest desulfurization efficiency in Thiobacillus.Sulfur can be accumulated in the extracellular and under anaerobic conditions,it can use nitrate as electron acceptor with the reduction of nitrate to N2 to realize the simultaneous removal of nitrogen and sulfur.It has many advantages over others and make itself an important engineering bacteria.There are two pathways of sulfur oxidation in Thiobacillus denitrificans,including SQR and Sox.When nitrate is used as an electron acceptor,sulfocompounds are oxidized mainly through SQR pathway.The SQR protein transfers the electron to quinone through the prothetic group FAD,the quinone is reduced to hydroquinone and the Sub-Sulphide(such as H2S)is oxidized to zero-valent sulfur.SQR protein is the first enzyme to access in the SQR pathway and is the only rate limiting enzyme in the SQR pathway.Therefore,the study of SQR protein is an important basis for the the improvement of Thiobacillus denitrificans strain.The results of previous studies indicate that the amphiphilic helix structure of C terminal can affect the binding of SQR protein to membrane and C terminal is important for forming part of the pathway providing access of the quinone substrate which is an important functional area.In this study,Kyte-Doolittle Hydropathy Plots?TMHMM?Amphipa Seek 1.3.5?Signal P 4.1?Tat P 1.0?PSORT?PSLpred were used to predict the hydrophobicity,transmembrane domain,signal peptide and intracellular localization of SQR protein and SQR protein is expected to be a peripheral membrane protein with no transmembrane domain and signal peptide.The homology modeling method was used to build SQR protein model and mutant model from Thiobacillus denitrificans ATCC25259 in Discovery Studio 3.5 platform.All models need to run the molecular mechanics and molecular dynamics simulation on GROMACS 5.1.2 to ensure that the protein model is in stable state with low energy.PROCHECK,Verify,Pro SA and ERRAT were used to evaluate the model and the results showed that the protein model of 3D has high rationality.Calculate interface interaction,amino acids,SAS and electrostatic potential distribution of the models.Compared with the wild type SQR protein,the number of hydrogen bonds and protein ligand contact area of No.7 mutant(Tyr411Gln Val412Lys)are increased.Structure changes most and the stability is improved.It is expected that enzyme activity of No.7 mutant(Tyr411Gln Val412Lys)will increase.Escherichia coli was used as a heterologous expression system to express SQR proteins.The constructed SQR and mutant expression vector were transfered into E.coli BL21(DE3)to express the protein with molecular weight of about 65 ku after adding IPTG.The purified protein contains His·tag which were purified by nickel column affinity chromatography.Proteins were detected by SDS-PAGE and Western Blot.Compared with the wild type SQR,the enzyme activity of No.7 mutant(Tyr411Gln Val412Lys)increased 23% while activities of the other mutants were decreased.Through simulation calculation and experimental verification,it is shown that the amphiphilic helix structure of C terminal of SQR has an important influence on the structural stability of protein.When the protein structure stability is reduced,the enzyme activity is decreased.At the same time,it also shows that homology modeling has a good guiding significance for the rational design of enzyme,which is helpful to select a good mutant.