Enzymatic Properties And Catalytic Mechanism Of Thio S-methyltransferase MddA From Bacteria

Dimethyl sulfur(DMS)is a volatile organic sulfur compound,which plays an important role in the global sulfur cycle and possibly affecting climate.It is the most abundant natural sulfide emitted into the atmosphere,the amount of sulfur transferred from the ocean to the atmosphere(in the form of DMS)is about 13-37 Tg per year.After DMS is released into the atmosphere,it could be oxidized.The oxidation products of DMS can act as cloud condensation nuclei which could affect the formation of clouds.Dimethylsulfoniopropionate(DMSP),an abundant organic sulfur compound,is the precursor of DMS in the oceans.Current studies have shown that DMS is mainly produced by microbial catabolism of DMSP in marine environments.In addition to the cleavage of DMSP,there are some microbial pathways independent of DMSP to produce DMS.Studies have shown that some marine and terrestrial aerobic bacteria can methylate methanethiol(MeSH)to produce DMS through a pathway called MeSH dependent DMS production(Mdd)pathway,and mddA gene has been found in many bacteria.MddA is a membrane bound S-adenosyl-L-methionine(SAM)-dependent methyltransferase,which methylates MeSH and produces DMS.In this study,MddA from Pseudomonas deceptionensis M1T strain was taken as the main research object.After heterologous expressed and purified,enzymatic properties and three-dimensional structure of MddA were analyzed.The process of of H2S methylation by MddA were studied.1.Sequence analysis,expression,purification and enzymatic properties of MddA,In this study,we synthesized the mddA gene from P.deceptionensis MIT and mddA-1,mddA-2,mddA-3 from three other different strains,and these four MddA proteins were expressed and purified.We studied MddA from P.deceptionensis MIT in detail,and determined its enzymatic properties and kinetic parameters.The results showed that the optimum temperature and pH of MddA were 30? and 8.0,respectively.The Km value of MddA for H2S was 0.41±0.04 mM,which was nearly one order of magnitude lower than the Km value of MeSH(1.99±0.11 mM).In addition,the kcat and kcat/Km values of MddA towards MeSH are both higher than those towards H2S,which means when H2S is used as the substrate,the consumption rate of the intermediate MeSH is higher than its production rate.Therefore,the intermediate MeSH will not accumulate notablely during these two steps of methylation.Compared with other thiol S-methyltransferases,it was found that MddA has relatively low Km value and relatively high catalytic efficiency towards H2S.The kinetic parameters of MddA towards several different substrates showed that the H2S is the natural substrate of MddA.2.Structure prediction and catalytic mechanism of MddAAlthough MddA could catalyze the methylation of H2S to produce DMS,its catalytic mechanism is unknown.By analyzing the predicted structure of MddA,we found six amino acid residues that may participate in the catalytic reaction,and constructed six corresponding site-directed mutants.Combined with structural analysis and biochemical verification,we proposed the possible molecular mechanism of MddA catalyzing the methylation of H2S.MddA catalyzes a bimolecular nucleophilic substitution reaction.In this reaction,H77 acts as a catalytic base to capture a proton from H2S,which makes H2S deprotonate and initiate electron transfer.After that,the sulfur atom of H2S would attack the methyl group of SAM,and the methyl group is transferred to the sulfur of H2S to form the intermediate product MeSH.After SAH is released from the catalytic cavity,SAM molecules enter the catalytic cavity again,and H77 is used as the catalytic base again to capture a proton from MeSH,which makes MeSH deprotonate and initiate electron transfer.After that,the sulfur atom of MeSH attacks the methyl group of SAM,and the methyl group is transferred to the sulfur of MeSH to form the final product DMS?The research in this chapter is of great significance to better understand the catalytic mechanism of MddA.In conclusion,we studied the enzymatic properties of MddA,and proposed the possible molecular mechanism of MddA catalyzing the methylation of H2S.The results provided important information for the further understanding of DMS production process.