Research Of Methyl Donor Diffusion Mechanism Of Ubig From E. Coli And Structural Studies Of SkgA From C. Crescentus

Part 1. The membrane association of UbiG regulates the entrance of methyl donor during the O-methyl transfer process for Coenzyme Q biosynthesisCoenzyme Q (Ubiquinone), an essential ingredient in the electron transport chain, is found in the inner mitochondrial membrane of eukaryotes as well as the plasma membrane of prokaryotes. Coenzyme Q plays a pivotal role in shuttling electrons from complex I or II to complex III for ATP synthesis in bacteria and higher eukaryotes. UbiG is a SAM-dependent O-methyltransferase, catalyzing two O-methyl transfer steps for ubiquinone biosynthesis in Escherichia coli. Our previous result identified that UbiG belongs to a novel class of membrane-binding proteins. Compared with the typical Class I SAM-dependent O-methyltransferase, UbiG possesses a unique sequence insertion between β4 and α10, which is used for membrane lipid interaction. Interestingly, this sequence insertion also covers the methyl donor binding pocket, and all attempts made to acquire the complex structure of UbiG with the methyl donor have been failed. Thus, the relationship between membrane binding and entrance of the methyl donor of UbiG during the O-methyl transfer process is a question that deserves further exploration.In this study, we firstly revealed that the membrane-binding region of UbiG gates the entrance of methyl donor. When bound with liposome, UbiG displays an enhanced binding ability toward the methyl donor product S-adenosylhomocysteine (SAH). We further employed protein engineering strategies to design UbiG mutants by truncating the membrane interacting region or making it more flexible. The isothermal titration calorimetry (ITC) results showed that the binding affinity of these mutants to SAH increases significantly compared with that of the wild-type UbiG. Moreover, we determined the crystal structure of UbiGΔ165-187 in complex with SAH. In light of this structure, we uncovered the methyl donor recognition model of UbiG. Collectively, our results provided a new angle to cognize the relationship between membrane binding and entrance of the methyl donor of UbiG, which is of benefit for better understanding the O-methyl transfer process for CoQ biosynthesis in vivo.Part II. Structural research of Caulobacter crescentus skgACaulobacter crescentus is a Gram-negative alpha-proteobacterium characterized by its unique asymmetric cell division pattern. There are two distinct cell form in its cell cycle with different morphologies and replicative potentials, replicating swarmer form and non-replicating stalker form, which requires a complicated cell cycle regulatory system controlling chromosomal replication and transcription spatially and temporally. There are two core signal pathways within the cell cycle regulatory system of C. crescentus:CckA-based phosphate transferring signal pathway and DivJ/PleC based DivK phosphorylation regulatory signal pathway. DnaA, CtrA, Ccrm and GcrA, four global regulators, dominate the regulation of the cell cycle of C. crescentus through controlling the expression of hundreds of downstream genes involved in cell cycle regulaton.SkgA is the first protein to be found as the starvation response regulator for H2O2 pressure. It is reported that SkgA performs function in promoting the expression of Catalase-peroxidase katG and the resistance of H2O2 pressure when facing carbon deficit. Based on the sequence alignment, SkgA belongs to MerR-like H-T-H transcription regulator family, thus SkgA is proposed to possess the similar function with other members of MerR-like regulator family. However, the research about SkgA is limited in the fields of starvation response regulation and the function of SkgA in transcription regulation remains unclear. It should be futher examined whether SkgA participates and what role it plays in cell cycle regulation.Here, we cloned, expressed, purified and crystalized the wild-type SkgA and SeMet-SkgA. Subsequently, we obtained the diffraction data of native SkgA and SeMet-SkgA at the resolution of 2.7A and 3.26A, respectively. Our work laid a foundation of following structural determination, and the structural research of SkgA is of benefit for further exploring its unknown function.