The Molecular And Kinetic Regulation Mechanisms Of Dimethylsulfoniopropionate(DMSP)Catabolism By Marine Roseobacter

Dimethylsulfoniopropionate?DMSP?,produced on a scale of 103 Tg a year by marine phytoplankton,microalgae and a few marine angiosperms,is a major participant in the global sulfur and carbon cycles.In some part of the ocean surface,DMSP can account for-10%of the fixed carbon.Microbial cleavage of DMSP is an important step in global sulfur and carbon cycles.Marine Roseobacter Clade?MRC?is one of the main microbial groups that catabolize DMSP.Microorganisms use DMSP lyases to cleave DMSP to generate volatile dimethyl sulfide?DMS?and acrylate.Volatile DMS is the bridge between the marine and the atmosphere sulphur pools and is an influential climate-affecting compound that can affect the global climate by enhancing the reflectivity of the earth to solar radiation.Acrylate is an important carbon source in marine environment.Many marine bacteria can metabolise acrylate as a sole carbon source for growth.It is of great significance to study the catabolic mechanism of DMSP.Research on it will bring better understandings on the global sulfiur and carbon circling and on the effect of the microbial metabolism to the environment.In this study,we took MRC as the research objects and mainly focused on the molecular mechanism of DMSP lyases,the evolutionary mechanism of DMSP lyases,the molecular mechanism of intracellular acrylate metabolism and the kinetic regulation mechanism of the DMSP catabolism.1.The molecular mechanism of microbial cleavage of DMSP to generate DMS by marine Roseobacter.Microbial cleavage of DMSP can produce about 300 million tons of DMS per year.There are various DMSP lyases.Eight different kinds of DMSP lyases have been discovered.However,there are limited researches on the molecular mechanism of DMSP lyases.DddP is the most widely distributed DMSP lyase in the ocean,which mainly exists in MRC.Here,we studied the molecular mechanism of RlDddP from Ruegeria lacuscaerulensis ITI₁157 to cleavage DMSP.We first validated the function of RlDddP by RT-qPCR and enzyme activity assays.The results showed that the expression of the R1dddP gene was dramatically induced by DMSP and the recombinant protein showed high DMSP cleavage activity.Then we detected the characterization of RlDddP.RlDddP showed an optimal pH of 6.0 and an optimal temperature of?60? for DMSP cleavage.The Km of RIDddP for DMSP cleavage was 17.1 ± 0.98 mM.Chelation agents EDTA and EGTA had little effect on the activity of RlDddP,whereas 1,1-o-Phenanthroline?OP?and 2,2-bipyridine can significantly inhibit the activity.Then the structures of RlDddP in complex with 2-?N-morpholino?ethanesulfonic acid,RlDddP in complex with phosphate,RIDddP mutant Y366A in complex with acrylate and RlDddP mutant D377A in complex with acrylate were solved.RlDddP is a dimer in solution.Each RlDddP monomer has a two-domain architecture and adopts a typical 'pitta-bread' fold structure.The two domains are positioned at an angle of approximately 90° to one another.The RlDddP dimer has two active centers.Each active center contains a two-iron cofactor and ten conserved residues.Among them,Asp377 is just beside the ?-C of DMSP and probably acts as the nucleophilic attack base in the reaction.Mutation on Asp377 leads to RlDddP inactive,demonstrating that Asp377 is the attack base.Then conformational alignment of the four structures was done and a 'iron-shift'phenomenon was found.This shift is a key step in the catalytic process,which can help stabilize the binding of DMSP and increase the acidity of the alpha hydrogen.Finally,based on the experimental results,we characterized a new ion-shift catalytic mechanism of RlDddP for DMSP cleavage.This study is of great significance for a better understanding of the release of DMS from DMSP cleavage.2.The evolutionary mechanism of DMSP lyases.Based on sequence and genome analyses,it is suggested that DMSP catabolism in bacteria evolves from the pre-existing catabolic pathways.DMSP lyases are various that eight known DMSP lyases belong to different families,indicating DMSP lyases have different evolutionary origins.In this study,we used RIDddP from R.lacuscaerulensis ITI₁157 as an example to study the evolutionary mechanism of it.Considering that DddP belongs to the M24 metallopeptidase family by sequence alignment,we firstly detected the protease function of RlDddP by RT-qPCR and enzyme activity assays.The expression of the R1dddP gene cannot be induced by peptide or casein and the recombinant protein shows no protease activity,demonstrating that RlDddP has completely lost the function of protease during the process that it evolves from a protease to a DMSP lyase.Then,we did a phylogenetic analysis of the DddPs.We found that DddPs formed a separate clade within the M24 metallopeptidase family and that the N-domains of DddPs may belong to a new domain family.The unique N-domains of DddPs are related to the formation of a small catalytic cavity which is specific for DMSP.Then,conformational alignment of the C-domains of RlDddP and M24 metallopeptidase was done.It showed that the residue that helps stable the intermediate in metallopeptidase was mutated to the aggressive base in RlDddP.The mutation of the key residue in the active center probably led to the loss of the protease catalytic capacity and the development of DMSP lyase catalytic capacity of RlDddP.Finally,based on all of the experimental results,we proposed an evolutionary mechanism for DddP.This study sheds light on the divergent evolution of DddP;leading to a better understanding of the marine bacterial DMSP catabolism and evolution of the DMSP lyases.3.The molecular mechanism of intracellular acrylate metabolism by marine Roseobacter.Except as an important carbon source in the marine environment,acrylate and its metabolism intermediate product acryloyl-CoA are toxic.Therefore,DMSP utilizing organisms require rapid metabolism system to detoxify acrylate and its metabolites.However,the molecular mechanism of acrylate remains unknown.Here,we studied the molecular mechanism of acrylate intracellular metabolism in MRC,a main microbial groups that catabolize DMSP.Firstly,we verified the acrylate metabolic pathways in MRC and found that it mainly used a PrpE-AcuI pathway to metabolize acrylate.Propionic acid coenzyme A ligase?PrpE?and enoyl coerunzyme A reductase?AcuI?are the key enzymes of the pathway.Then the structures of PrpE from Dinoroseobacter shibae DFL 12 and AcuI from R.pomeroyi DSS-3 were solved.PrpE is a monomer in solution.The topological structure of PrpE is similar to those of other acyl-or aryl-CoA synthetases.It contains two domains.Acul is a dimer in solution.The structure of Acul resembles that of YhdH?PDB code:3NX4?from Escherichia coli and it contains a typical rossmann fold which binds the NADPH cofactor.Then,we studied the function of the conserved residues in the active center of PrpE and Acul.PrpE possesses two conformations,adenylate-forming conformation and thioester-forming conformation.Two highly conserved residues in PrpE,Lys588 and Gly502,located at the active center in the two conformations,respectively.Mutations on these residues lead to PrpE inactive.Thus,Lys588 and Gly502 are probably the key residues in the catalytic mechanism of PrpE,which Lys588 is crucial for the adenylate-forming half-reaction and Gly502 is crucial for the thioester-forming half-reaction.In AcuI,there are several hydrophilic residues around the bounded NADPH.Among them,only mutation on Arg323 significantly affects the activity of Acul.Thus,Arg323 probably acts as the br???nsted acid in the catalytic process of Acul and NADPH probably acts as the reducing agent to provide reducing power.Finally,based on the experimental results,we proposed a molecular mechanism of acrylate intracellular metabolism,which will provide a better understanding of the acrylate intracellular metabolism.4.A kinetic regulation mechanism of the DMSP catabolism in marine Roseobacter.The catabolic metabolism of DMSP is a complicated process that involves many steps,and need a variety of enzymes function cooperatively.Except as an important participant of the global sulfur and carbon cycles,DMSP also has important physiological functions.DMSP-catabolizing bacteria usually accumulate DMSP as osmolyte,antioxidant and antifreeze.The intracellular concentration of DMSP can reach at the millimole level.In addition,acrylate and acryloyl-CoA are toxic.So microorganisms,especially DMSP-catabolizing bacteria,need a system to swiftly metabolise and detoxify these metabolites.However,so far,there is no study involved in the regulation of DMSP catabolism.Here,we studied the kinetic regulation mechanism of DMSP cleavage process of MRC.Firstly,we compared the Km values of PrpE and Acul with various kinds of DMSP lyases and found that in most cases,it decreased in the order DMSP lyases>PrpEs>>AcuIs.Then we compared the kcat/Km values of PrpE and AcuI with various kinds of DMSP lyases and found that in most cases,it increased in the order DMSP lyases<PrpEs<<AcuIs.Based on these,we proposed a kinetic regulation mechanism of the DMSP cleavage process.The differences in the efficiency and substrate affinities of the enzymes guarantee the intracellular accumulation of DMSP and the rapid metabolism of acrylate and acryloyl-CoA.Then we detected the abundances of PrpE and Acul in nature.It was found that PrpE and Acul were not only present in MRC,but also in various organisms across all domains of life.Besides,in addition to DMSP catabolism,acrylate/acryloyl-CoA is probably produced through other metabolisms,such as lactate,propionate,beta-alanine and glucose metabolism.Thus,our kinetic regulation model may be extended to other organisms and is likely relevant to many other metabolic processes and environments above just DMSP catabolism in marine environments.In conclusion,this study sheds light on the molecular mechanism of DMSP lyases,the evolutionary mechanism of DMSP lyases,the molecular mechanism of intracellular acrylate metabolism and the kinetic regulation mechanism of the DMSP catabolism of MRC,leading to a better understanding for marine bacterial DMSP catabolism and the global sulfur and carbon cycles.