Structural And Functional Analyses Of The Galactonate Transcription Repressor DgoR From Escherichia Coli

In human and animal intestines,there are a variety of intestinal microfloppies,which use intestinal nutrition to grow and multiply,and form a mutually beneficial intestinal microbial ecosystem.Modern biological and medical studies have found that intestinal microbes are associated with a variety of diseases.Meanwhile,the theories of lung-intestinal axis,brain-intestinal axis and liver-intestinal axis,which have developed rapidly in recent years,also indicate that intestinal microbes have many connections with traditional Chinese medicine theories.In-depth research on the microecology of intestinal flora is becoming"a golden key to open the door of TCM".As an important part of gastrointestinal microfloppies,Escherichia coli can cause a variety of diseases under certain conditions,and even death in severe cases.Studies have shown that D-galactoic acid is a widespread sugar acid,and its metabolism is considered to be important for intestinal colonization of Escherichia coli,and can serve as an important signal to induce virulence factor synthesis.Dgo operon of D-galactonate metabolic pathway in EScherichia coli is mainly regulated by transcriptional regulator DgoR protein.DgoR belongs to FadR family protein of GntR superfamily,and most FadR family members are involved in the transcriptional regulation of substrate oxidase in amino acid metabolism.Although some of the transcriptional regulators of the FadR family have been well described at the molecular level,the binding bag of DgoR to D-galactonate and its substrate specificity have not been well studied.Based on the close relationship between intestinal microorganisms and traditional Chinese medicine theory,the importance of glucose and acid metabolism for microbial growth,and the important role of D-galactonate in bacterial colonization and toxicity,it is necessary to further study the transcriptional regulatory factors in D-galactonate metabolism pathway of bacteria.In this study,the DgoR protein of D-galactonate transcription regulator of Escherichia coli was purified by molecular cloning and expression,and the crystal structure of DgoR protein was analyzed by X-ray crystal diffraction,and the gene site-directed mutation,gel migration(EMSA)and microthermal activity(MST)experiments were performed.The binding pocket of escherichia coli derived transcriptional regulatory factor DgoR and substrate D-galactonate was verified,and the results were as follows:1.The C-terminal domain(EcoDgoR-C)of EcoDgoR was crystallized in this study,and the crystal structure of EcoDgoR-C was successfully obtained with 2.2 A resolution.Based on the GntR superfamily members,the GntR superfamily members had similar helix-turn-helix(wHTH)DNA at the N-terminal(NTD)Binding domain was modeled by Tm0439(PDB:3FMS)DNA binding domain structure,and the overall structure model of DgoR was established.2.Based on the crystal structure,a divalent metal ion(Zn2+)was found in the EcoDgoR-D-galactonate binding bag,which was treated by EDTA The important role of divalent metal ions in the binding of EcoDgoR to D-galactonate was confirmed by readding divalent metal ions.The absence of divalent metal ions will greatly reduce the ability of EcoDgoR to bind D-galactonate.3.The molecular docking of EcoDgoR and D-galactonate was carried out to propose the key amino acid residues that may be involved in the interaction between EcoDgoR and D-galactonate,and the EMSA、MST method was used to determine the R102 Q173 R224 T191 in the EcoDgoR-D-galactonate binding bag.H150 and H195 are crucial for DNA and D-hemilactic binding,while S221 W181 and R179 are important for D-galactonate binding,but have little influence on DNA binding.4.In amino acids bound to divalent metal ions,mutations in H150 and H195 will result The binding ability of EcoDgoR and D-galactonate is extremely reduced,which may be related to the instability of binding between EcoDgoR and divalent metal ions caused by mutations of H150 and H195,while D146 mutation has no significant influence on the binding of divalent metal ions.5.Based on the many links between intestinal microflora microecology and traditional Chinese medicine theories,this study revealed the mechanism of DgoR binding operon and D-galactonate at the molecular level,which will provide theoretical ideas for further exploring the small molecules of Traditional Chinese medicine that inhibit the utilization of D-galactonate by Escherichia coli in Traditional Chinese medicine.