Arabidopsis Mercaptopyruvate Sulphurtransferase Has A New Function Of Catalyzing The Production Of H₂S

Hydrogen sulfide(H2S)is a new member of the gasotrainsmitters family including nitric oxide(NO)and carbon monoxide(CO),and has received extensive attention and systematic research in recent years.H2 S can not only participate in a variety of physiological and pathological processes in animals,but also have the effect of promoting plant growth and enhancing plant abiotic stress resistance.H2 S is produced in vivo mainly through some enzymatic reactions.In animals,three enzymes have been found to be involved in the production of endogenous H2 S,namely cystathionine ?-lyase(CSE)?cystathionine ?-synthase(CBS)and mercaptopyruvate sulphurtransferase(MST).In plants,there are four types of enzymes that can catalyze the emission of endogenous H2 S,namely cysteine desulphydrases(CDes)?cysteine desulfurases??-cyano-alanine cynthase(CAS)and O-acetylserine(thiol)lyase(OASTL).Among them,CBS,CSE,CDes,CAS,OASTL,and cysteine desulfurase all catalyze the production of H2 S in plants and animals using cysteine(Cys)as substrates.Their enzymatic properties and the specificity of substrate have been systematically studied.In animals,MST has both the function of removing cyanide and catalyzing the production of H2 S.Its substrate is mercaptopyruvic acid sodium salt.At present,there are many studies on the function of At MST in plants,which are mainly considered that it is an important enzymes to removal of cyanide in plants.There is no report on whether At MST in plant can catalyze the production of H2 S.If At MST can catalyze release of H2 S in plants,it is undoubtedly an important supplement to the research field of gasotrainsmitters H2 S.In this paper,the relationship between MST and the production of H2 S in Arabidopsis thaliana was analyzed using prokaryotic expression and in vitro purification.The results are as follows:1.Construction of Expression Vectors: At MST(At1g79230)gene was obtained by comparison with the NCBI website.It showed that this gene had three transcripts,named At MST.1,At MST.2 and At MST.3.The three transcripts were cloned using Arabidopsis c DNA as a template,and the At MST.2 gene was failed to cloned.Other target genes were ligated into the prokaryotic expression vector p ET28 a to obtain the recombinant plasmid p ET28a-At MST,which was correctly identified by sequencing and successfully transformed into E.coli BL21(DE3)competent cells.2.Optimum conditions for inducing protein expression: The successfully transformed E.coli BL21(DE3)/p ET28a-At MST was induced by isopropyl-?-D thiogalactoside(IPTG)and expressed two proteins At MST.1 and At MST.3,which molecular weights were 37.9 k D and 32.2 k D.The optimal induction conditions were as follows: the final concentration of IPTG was 0.05 mmol·l-1,and the culture was induced at 20°C for 20 h.3.Optimization of protein purification conditions: The above successfully induced proteins were purified by Ni Sepharose column,and the At MST.1 protein with a molecular weight of 37.9 k D was purified.After screening,the best purification conditions were determined as: 20 mmol·l-1 imidazole to disrupt the bacteria cells,the protein impurity was eluted with 50 mmol·l-1 imidazole and the target protein was eluted with 300 mmol·l-1 imidazole.A single protein band of interest was then identified by SDS-PAGE.The 32.2 k D At MST.3 protein was not purified successfully.4.Enzyme activity assay: The efficiency of the purified At MST.1 protein to produce H2 S was measured using mercaptopyruvic acid sodium salt as a substrate.The results showed that it had the activity to catalyze the production of H2S;as for the unsuccessfully purified protein At MST.3,the detection of H2 S was to use the soluble protein solution extracted in the cell lysis buffer of bacteria with Mercaptopyruvic acid sodium as a substrate,and it demonstrated that this protein can also catalyze the production of H2 S.In summary,this study constructed the prokaryotic expression vector,purified the protein and measured the enzyme activity,the results suggested that Arabidopsis MST has a new function of catalyzing the production of H2 S with mercaptopyruvic acid sodium salt as a substrate.