The Structures And Catalytic Mechanisms Of Key Enzybmes Involved In The Catabolism Of Dimethylsulfoniopropionate Metabolites Aicryloyl-CoA And Dimethylsulfide In Marine Bacteria

Dimethylsulfo niopropio nate(DMSP)is an important organic sulfur co npo und in the oceans.It is one of the most abundant forms of organic sulfur on the earth.DMSP can be produced on a scale of 103 Tg each year in global oceans.DMSP can be cleaved by DMSP lyase to produce dimethylsulfide(DMS)and acrylate in microorganisms.DMS,a volatile organosulfur compound,is considered to play an important role in the global sulfur cycle.It can enter the atmosphere through sea-to-air emission and is the main form of sulfur released from the ocean into the atmosphere.In addition,DMS also plays a role in the formation of cloud condensation nuclei and can affect the formation of clouds.The synthetic and metabolic pathways of DMS have raised widespread attention.Another metabolite of DMSP cleavage is acrylate.Acrylate can be used as a carbon source for DMSP-catabolizing bacteria in the oceans.The metabolite of acrylate is acryloyl-CoA,which is toxic to cells.The accumulation of acryloyl-CoA in cells will serio usly affect the physiological and biochemical status of the organisms.Thus,the metabolic pathways of acryloyl-CoA also have received extensive attention The results of this dissertation are as follows:(1)Crystal structure and catalytic mechanism of acryloyl-CoA reductase AculAfter transported into marine bacterial cells,DMSP is cleaved by DMSP lyases,producing DMS and acrylate.Acrylate can be further metabolized as a carbon source in cells.Acrylate can be metabolized to acryloyl-CoA by propionate-CoA ligase PrpE or acyl-CoA transferase AcuN.Acryloyl-CoA is toxic to bacterial cells.It cannot accumulate in cells and must be metabolized.Acryloyl-CoA can be metabolized to propionyl-CoA by acryloyl-CoA reductase AcuI,or be metabolized to 3-hydroxypropionyl-CoA(3HP-CoA)by acryloyl-CoA hydratase AcuH,thus preventing the accumulation of acryloyl-CoA.The structure and catalytic mechanism of the key enzymes PrpE and AcuN involved in the metabolism of acrylate have been studied in our laboratory.On this basis,in order to elucidate the molecular mechanism of acryloyl-CoA metabolism in marine bacteria,the structure and catalytic mechanism of acryloyl-CoA reductase AcuI and acryloyl-CoA hydratase AcuH were studied.AcuI can reduce acryloyl-CoA to propionyl-CoA,which plays a role in the metabolism of acryloyl-CoA.Here,we took the AcuI from R.pomeroyi DSS-3,the model strain of marine Roseobacter clade,as our research target and studied the crystal structure and catalytic mechanism of acryloyl-CoA reductase AcuI.AcuI was expressed,purified and crystallized.The structure of AcuI without any ligand(apo-Acul)and the structure with NADPH(AcuI-NADPH)were solved.The root mean square deviation(RMSD)between the two structures is 0.313 A,which indicates that the three-dime nsio nal structure of AcuI has few conformational changes after NADPH is bound.Each AcuI monomer has two domains,the catalytic domain and the Rossmann fold domain.The results of enzymatic activity assay showed that the activity decreased significantly after Arg323 was mutated to alanine.Similarly,when Arg323 was mutated to uncharged isoleucine,the enzymatic activity was almo st completely abolished.However,the enzymatic activity was almost unchanged when Arg323 was mutated to lysine,a similar basic residue.These results suggest that Arg323 may participate in electron transfer and act as proton donor in the catalytic process.Based on these results,the catalytic mechanism of acryloyl-CoA reduction catalyzed by AcuI was proposed.In this reaction,the hydrogen of NADPH nicotinamide C4 would attack the C3 of acryloyl-CoA and transfer an electron,then form an enolate intermediate.The enolate intermediate then transfer the electron to Arg323 and finally forms the product propionate-CoA.In addition,it was found that most of the strains containing PrpE also contain AcuI,further indicating that Acul plays an important role in the metabolism of acryloyl-CoA.These results are of great significance for better understanding of the metabolism of acryloyl-CoA.(2)Crystal structure and catalytic mechanism of acryloyl-CoA hydrataseRdAcuHAcuH is an enoyl-CoA hydrase that catalyzes the hydration of acryloyl-CoA to 3-hydroxypropionyl-CoA(3-HP-CoA).It plays a role in the metabolism of acryloyl-CoA.With the AcuH from strain R nubinhibens ISM(RdAcuH)as our research object,the crystal structure and the catalytic mechanism of RdAcuH for acryloyl-CoA hydration were studied.RdAcuH was expressed,purified and crystallized.The overall structure of RdAcuH is a hexamer(a dimer of trimers)in the asymmetric unit.Each RdAcuH monomer contains two domains,one is the N-terminal domain(N TD)and the other is the C-terminal domain(CTD).The active center is located between the NTD of a monomer and the CTD of an adjacent monomer.The structure of RdAcuH is very similar to that of enoyl-CoA hydratase ECH from Rattus norvegicus.By sequence alignment and structure superposition,two conserved residues in RdAcuH,Glu112 and Glu132,were found.The enzymatic activities of E112A and E132A mutants are not detected,indicating that Glu112 and Glu132 play important roles in the catalytic reaction of RdAcuH.Based on the analysis of RdAcuH structure and biochemical experiments,the catalytic mechanism of RdAcuH was proposed.In the reaction,Glu132 attacks a catalytic water molecule and activate the water molecule.The activated water molecule then attacks the C3 of acryloyl-CoA to form an intermediate state.After that,acryloyl-CoA sends the redundant electron back to Glu132,and the water molecule is added to acryloyl-CoA to form the product 3-HP-CoA.RdAcuH can catalyze the hydration of acryloyl-CoA.In addition,RdAcuH can also catalyze the hydration of 3-methylthioacryloyl-CoA(MTA-CoA),a downstream metabolite in DMSP demethylation pathway.Homologues of RdAcuH are widespread and can be found in many strains that cannot metabolize DMSP,which indicate s that the catalytic mechanism of acryloyl-CoA by RdAcuH may be extended to other metabolic processes and have a wider significance.(3)Crystallization and structural analysis of DMS monooxygenase DmoADMS is a volatile organic sulfide compound and it is the main form of natural sea-to-air emission of sulfur.Current studies on DMS metabolism have shown that biodegradation is the main degradation mode of DMS.DmoA is a DMS monooxygenase that can catalyze the metabolism of DMS to methanethiol.Here,the crystal structure of DMS monooxygenase DmoA from strain Hyphomicrobium sulfonivorans was studied.DmoA was expressed,purified and crystallized.The asymmetric unit contains two DmoA monomers.DmoA monomer consists of the TIM-barrel structure and the additional insertion(AI)structure.There are five AIs(AI1,AI2,AI3,AI4 and AI5)and they are all located between the outer a-helices and the inner ?-strands.This kind of additional insertions were also found in the structure of long-chain alkane monooxygenase LadA and dibenzothiophene sulfone monooxygenase BdsA.The structures of DmoA,LadA and BdsA were compared and their monomic structures are similar.We analyzed the three structures and found that the substrate binding pocket of DmoA is smaller than those of LadA and BdsA.The smaller substrate binding pocket of DmoA is consistent with its smaller substrate(DMS).The changes of substrate binding pockets are mainly caused by the differences of AI5?3 and AI3 in these three structures.These results would provide important informations for better understanding of the microbial degradation process of DMS.(4)Expression and enzymatic properties of methanethiol methyltransferase MddADMS is an important gaseous organic sulfur.At present,it is widely considered that DMS is mainly produced by the cleavage of DMSP in the oceans.However,so me studies indicated that in marine,freshwater and terrestrial environments,there are still pathways to generate DMS without DMSP.A DMSP-independent DMS production pathway was found in Pseudomonas deceptionensis M1T,a strain isolated from Antarctic marine sediment.This pathway is known as methanethiol-dependent DMS production pathway in which methanethiol is transferred into DMS by methyltransferase MddA.MddA is a key enzyme in this pathway.MddA is a membrane protein,its expression,purification and biochemical properties have not been reported yet.The MddA from P.deceptionensis M1T was used as our research object.The expression system in Escherichia coli was established and MddA with in vitro activity was purified.By screening the expression vectors and strains,we established the best expression system of MddA using the pET-15b vector and E.coli C43(DE3).In addition,Detergents were screened and n-Dodecyl-?-D-maltopyranoside(DDM)was chosen as the most suitable detergent.After optimizing the conditions,the stable and homogeneous MddA protein was obtained.Then the biochemical properties of MddA were analyzed.Using DDM as a detergent,the optimum pH and temperature for MddA reaction were 8.0 and 40?,respectively.These results provided an important foundation for further researches on the crystal structure and catalytic mechanism of MddA.In this dissertation,the structures and catalytic mechanisms of key enzymes AcuI and RdAcuH in the metabolic process of acryloyl-CoA,the structure of key enzyme DmoA in the metabolic process of DMS and the enzymatic properties of key enzyme MddA in the synthesis process of DMS were studied.The results of this study provide important information for a better understanding of the metabolic process of DMSP.