The Catalytic Mechanisms Of Key Enzymes Involved In The Demethylation Pathway Of Dimethylsulfoniopropionate(DMSP)Catabolism And The Kinetic Regulation Mechanism Of This Pathway In Marine Roseobacter

Dimethylsulfoniopropionate(DMSP)is a major participant in marine carbon and sulfur cycles.DMSP is produced in earth's surface oceans to petagram levels annually by marine phytoplankton,macroalgae and bacteria,and the production may account for as much as 10%of the total carbon fixation in some area of the ocean surface.DMSP has many different physiological functions.As an important sulfur and carbon source,environmental DMSP can be utilized by taxonomically diverse bacteria,among which the Roseobacter and SAR11 clade are the most prominent members.Marine bacteria catabolize DMSP via two pathways,including the cleavage pathway and the demethylation pathway.While only about 10%of DMSP in environmental is routed through the cleavage pathway,the majority of DMSP is thought to be catabolized via the demethylation pathway.In the cleavage pathway,DMSP is cleaved by lyases to generate dimethyl sulfide andacrylate.Acrylate is further metabolized by PrpE and AcuI,and then be utilized by bacteria.In the demethylation pathway,DMSP is catabolized by four enzymes(DmdA,DmdB,DmdC and DmdD/AcuH)to generate methanethiol,which then be utilized by bacteria.The demethylation pathway in marine bacteria is reported to be responsible for the majority of environmental DMSP.Studying the crystal structures and the catalytic mechanisms of key enzymes and the kinetic regulation mechanism of DMSP catabolism in the demethylation pathway is helpful to understand the catabolism of DMSP and the role of marine microbial activities in promoting the global carbon and sulfur cycles.The crystal structures and the catalytic mechanisms of DmdA and DmdD/AcuH have been reported.In this dissertation,we took marine Roseobacter clade as the objects and mainly focued on the structures and catalytic molecular mechanisms of DmdB and DmdC and the kinetic regulation mechanism of the DMSP catabolism in the demethylation pathway by Roseobacter.1.The sturcture and the catalytic molecular mechanism of MMPA-CoA ligase in marine RoseobacterThe key enzyme DmdB in the demethylation pathway of marine bacteria is a 3-methylmercaptopropionate(MMPA)-Coenzyme A(CoA)ligase,which catalyzes the ligation of MMPA,the product of the DMSP demethylase DmdA in the demethylation pathway,and CoA to generate MMPA-CoA.We studied the structure and catalytic mechanism of DmdB from Ruegeria lacuscaerulensis ITI-1157.We firstly analyzed the function of dmdB by RT-qPCR and the enzyme activity assays.Results showed that the expression of dmdB can be up-regulated by DMSP and the recombinant DmdB protein showed high MMPA-CoA ligation activity.Then we characterized DmdB.Gel filtration analysis indicated that DmdB is a dimer in solution.Then we solved the crystal structures of DmdB in complex with ADP,the competitive inhibitors of ATP,and the DmdB mutant Lys523A1a in complex with AMP and MMPA.DmdB is a dimer.Each monomer consists of a large N-terminal domain and a small C-terminal domain,and the active center is located at the interface between the two domains.DmdB goes through twice conformational changes in the catalytic reaction.DmdB adopts the open conformation and the binding of ATP promotes the 64° rotation of the C-terminal domain of DmdB,leading to the first conformational change to form the adenylate-forming conformation,under which DmdB catalyzes the first step reaction.Based on structural analysis,mutation asssy verification and circular dichroism spectrum analysis,we found that Lys523 is an important catalytic residue.Then we systematically studied the functions of several conserved residues in the catalytic center and found the key residues involved in substrate binding and catalysis.We analyzed the thioester-forming conformation of DmdB and the function of key residues based on structural alignment analysis between DmdB and other reported acyl-CoA ligase.The result showed that the C-terminal domain of DmdB will rotates,leading to the second conformational change of DmdB to form the thioester-forming conformation.Finally,based on all of the experimental results,we proposed the molecular mechanism of the ligation of MMPA and CoA to generate MMPA-CoA by DmdB catalysis.Multi-sequence alignment analysis suggested that the proposed catalytic mechanism of DmdB has universal applicability in marine bacteria.This study is of great significance to better understand the catabolism of DMSP by marine bacteria through the demethylation pathway.2.The structure and the catalytic molecular mechanism of MMPA-CoA dehydrogenase DmdCIn the demethylation pathway,MMPA-CoA dehydrogenase DmdC with FAD as a cofactor catalyzes MMPA-CoA,the product of upstream enzyme DmdB,to generate methylthioacrylyl-CoA(MTA-CoA).We studied the structure and catalytic mechanism of DmdC from Roseovarius nubinhibens ISM.We firstly analyzed the function of dmdC by RT-qPCR and the enzyme activity assays and characterized DmdC.Then we solved the crystal structure of DmdC.Structural and gel filtration analysis showed that DmdC is a dimer,and the two monomers of DmdC are assembled through a large interface.Based on molecular docking,we predicted the FAD binding site and the function of key residues in the catalytic center.We then identified the residues that involved in FAD binding and catalysis.Based on the structural alignment analysis between DmdC and other reported acyl-CoA dehydrogenases,we analyzed the residues that are involved in MMPA-CoA binding and catalysis.Finally,based on the experimental results,we proposed the molecular mechanism of the dehydrogenation of MMPA-CoA to generate MTA-CoA by DmdC catalysis.Sequence alignment analysis shows that the proposed catalytic mechanism is likely universally adopted by different bacterial DmdCs.This study is of great significance to further understand the demethylation pathway of DMSP catabolism in marine bacteria.3.The kinetic regulation mechanism of the demethylation pathway of DMSP catabolism in marine RoseobacterThe catabolism of DMSP in marine bacteria is a complicated process.The cooperation among enzymes involved in the cleavage pathway and the demethylation pathway accomplishes the catabolism of DMSP and guarantees its physiological functions.The concentration of DMSP in seawater is very low,which is at nanomolar level.After enrichment,the intracellular concentration of DMSP in bacteria cells can reach millimolar level.Researchers have revealed the kinetic regulation mechanism of DMSP catabolism in the cleavage pathway.In this dissertation,we studied the kinetic regulation mechanism of DMSP catabolism in the demethylation pathway.We firstly verified the functions of four genes in the demethylation pathway by RT-qPCR and the enzyme activity assays.Then we detected the Km values of the four enzymes and analyzed their affinities.Results showed that the Km values of DmdAs from different bacteria are at ten millimoles level,which are the same as those of DMSP lyases,indicating that they all have low affinities to DMSP.The low affinities of those enzymes ensure the high intracellular concentration of DMSP to exercise its physiological functions.Just like the Km value of propionic acid CoA ligase PrpE of the cleavage pathway,the Km values of DmdB and DmdC in the demethylation pathway are at millimolar levels,and are lower than that of DmdA,indicating that the affinities of DmdB and DmdC to their substrates are higher than that of DmdA.The increase of substrate affinity ensures the continued metabolism and is benefit for substance transformation and energy flow.MTA-CoA hydratase DmdD is the last enzyme in the demethylation pathway.Its Km value is at micromolar level and its catalytic efficiency is very high.The high affinity and catalytic efficiency of DmdD ensure the rapid metabolism of its substrate to avoid their accumulation in cells,which is the same as the reported AcuI and also implies the cytotoxicity of MTA-CoA.Based on our analyses,we proposed a kinetic regulation mechanism of DMSP catabolism in the demethylation pathway.The difference in the substrate affinities of DmdA,DmdB,DmdC and DmdD guarantees the intracellular accumulation of DMSP for its physiological functions and the rapid metabolism of the toxic MTA-CoA to accomplish DMSP catabolism.This dissertation sheds light on the crystal structures and the catalytic mechanisms of two key enzymes DmdB and DmdC and the kinetic regulation mechanism of DMSP catabolism in the demethylation pathway.Our study is helpful for better understanding DMSP catabolism and the global sulfur and carbon cycles.4.Other results:optimization of the small and pilot scale fermentation conditions for the elastase myroilysin production by the deep-sea bacterium Myroides profundi D25In this dissertation,after systematically studying the organic sulfur DMSP catabolism by marine bacteria,we also studied the fermentation processe of deep-sea bacteria for protease production.Previous studies have shown that elastase myroilysin is the most abundant protease secreted by deep-sea sedimental bacterium M.profundi D25,which has a strong collagen-swelling ability,suggesting its promising biotechnological potential.Because myroilysin cannot be maturely expressed in Escherichia coli,it is important to improve the production of myroilysin in the wild strain M.profundi D25.In this dissertation,we firstly optimized the culture conditions of strain M.profundi D25 for myroilysin production by using single factor experiments.Under the optimized conditions,we conducted several small and piolt scale fermentations of M.profundi D25 and successfully set up the small and pilot scale fermentation processes of M.profundi D25 for myroilysin production,which lays a foundation for the industrial production of myroilysin and is helpful to develop the application of myroliysin in the biotechnological field.