Structural biology and analytical chemistry approaches for characterizing C-glycoside metabolic enzymes in human gut microbiota.Protocol. One, protein production and purification. Construction of expression vector.
Obtain the gene cluster sequence GenBank LC422372.1 from NCBI. Clone the genes into a modified pET-28a vector. Transform the plasmids into E.coli BL21 DE3 competent cells.
Subsequently culture the bacteria in LB medium with 50 micrograms per milliliter kanamycin at 37 degrees. Add 0.2 millimoles IPTG to the medium when the bacterial density OD600 reaches 0.6. Culture the bacteria at 16 degrees for 16 hours.
Centrifuge the bacterial culture at 4, 000 times g at four degrees. Discard the supernatant and retain the bacterial pellet. Resuspend the bacterial pellet in Buffer A.Purification of protein.
Add one millimole PMSF to the bacterial resuspension. Lyse the bacteria suspension by an ultrasonic cell breaker. Centrifuge the bacterial lysate at 48, 000 times g for 40 minutes at four degrees.
Purify the protein in the supernatant using the Nickel affinity chromatography NTA column. Elute the bound protein with Buffer B in a gradient. Collect samples with high UV 280 nanometer absorption and clear protein bands from SDS-PAGE for the next step.
Dialyze the samples against Buffer C.Purify the dialyzed protein using an ion exchange chromatography column. Bind the target protein to the column with Buffer C.Elute the bound protein with Buffer D ingredient. Purify the protein using a size-exclusion column with Buffer E.Collect protein samples for the next step.
Concentrate the protein samples to 20 milligrams per milliliter, aliquot it, rapidly freeze it with liquid nitrogen. Store it at 80 degrees for future use. Measure the protein concentration using QuickDrop with UV 280 nanometer.
Circular dichroism, CD, spectrum data collection. Dilute the proteins to 0.2 milligrams per milliliter in one times PBS buffer, pH 7.4. Set the instrument scanning speed to 50 nanometer per minute and the spectral bandwidth to one nanometer.
Collect CD spectral data in the wavelength range of 200 to 260 nanometer. Subtract the PBS background and take the average value from three measurements. Two, protein crystallography and structured determination.
Protein crystal growth and optimization. Perform crystallization initial screening with sitting-drop method using the protein crystallization kits on 48-well crystal plates. Set the precipitant and protein concentration gradients to optimize protein crystallization using the hanging-drop method.
Transfer crystals to a soaking solution containing reservoir buffer and target compounds at 16 degrees for 30 minutes after complete growth. Transfer crystals to a cryoprotectant solution containing crystallization buffer supplemented 25%glycerol and immediately stored in liquid nitrogen for data collection. Data collection and structured determination of protein crystals.
Collect the complete set of X-ray diffraction data at the beamlines of the SSRF of the NFPS using an incident wavelengths of 0.978 angstroms. Process crystal diffraction data initially using the program crystallographic data processing software. Carry out phasing using an automatic data processing program.
Use crystal structure determination software for automatic structure model refinement and optimization. Three, reaction activity monitoring and determination of kinetic parameters. Monitoring of DgpA/B/C activity by HPLC.
Assemble the C-glycoside cleavage reaction system in PBS buffer, pH 7.4, containing 20 micromoles per milliliter DgpA/B/C, one millimoles per liter manganese ion, one millimoles per liter NAD, 10 millimoles per liter DTT, and 0.1 millimoles per liter substrate, puerarin, daidzin, genistin. Mix thoroughly by pipetting and incubate at 37 degrees for eight hours. Add three times the amount of methanol to the reaction system to terminate the reaction.
Centrifuge the reaction solution at 16, 000 times g for 15 minutes to remove the precipitate. Filter the supernatant using a 0.22 micrometer filter membrane. Perform HPLC analysis with gradient elution at a flow rate of one milliliter per minute, detection wavelengths of UV 265 nanometer, and an injection volume of 10 microliters.
Monitoring of DgpA activity with 2, 6-dichlorophenol indophenol, DCPIP, assay. Incubate 0.8 millimoles per liter DCPIP, 50 micromole per liter protein, and 10 millimoles per liter substrate in one times PBS buffer, pH 7.4. After 20 hours of reaction, record the absorbance change of DCPIP at 600 nanometer using a microplate reader.
Determination of inhibition rate of glucose on the C-glycosidic bond cleavage reaction of puerarin. Carry out the reaction in one times PBS buffer, pH 7.4, at different concentrations of glucose to the reaction system as described in section 3.1. Perform the reaction at 37 degrees for 12 hours.
Perform quantitative analysis of the generated products using HPLC. Determination of kinetic parameters. The concentration of puerarin is set from 0.01 to four millimoles per liter, and that of daidzin and genistin was from 0.01 to two millimoles per liter in the DgpA/B/C C-glycoside cleavage reaction.
Carry out the reaction at 37 degrees for eight hours according to the method described in section 3.1. Derive the Km and kcat kinetic parameters by fitting the calculated reaction rate and substrate concentration to a Michaelise-Menten model in Prism. Four, preparation and detection of reaction intermediate product.
Preparation of reaction intermediate product. Use daidzin and genistin as substrates to carry out the reaction according to the methods described in section 3.1 with a reaction system of 90 milliliters. Purify the intermediate product using a preparative HPLC with a preparative liquid chromatography column.
Dry the purified intermediate products with a vacuum centrifugal concentrator for future use. Characterization of reaction intermediate product with LC-MS. Dissolve a portion of the purified intermediate products from the daidzin and genistin reactions in methanol to prepare solutions of each compound at 50 micrograms per milliliter.
Centrifuge at 13, 500 times g for 10 minutes and collect the uspernatants for LC-MS/MS analysis. Use the same gradient elution program as in the HPLC analysis for LC-MS analysis with an injection volume of five microliter. Characterization of reaction intermediate product with NMR.
Dissolve a portion of the purified intermediate products from the daidzin reaction in dimethyl sulfoxide-deuterated six for proton and carbon 13 NMR spectroscopy. Record the NMR spectra on a NMR instrument at 400 megahertz for a proton. Record the NMR spectra on a NMR instrument at 175 megahertz for carbon 13.
Representative results. Overall, we designed a combined experimental approach that includes protein production and purification, crystallization experiments, activity assays, and the identification of reaction product structures in figure one. Specifically, we obtained high-purity proteins through a three-step purification chromatography, ion exchange chromatography, and gel filtration chromatography in figure two.
In the crystallization experiments, we determined the crystal structures of DgpA, and the DgpB/C complex with resolutions of 2.7 angstroms and 2.1 angstroms, respectively, in figure 3A to D.We further discovered that the DgpA adopts in auto-inhibited confirmation in its hexameric form in figure 3B and C, which was validated using the DCPIP reduction assay in figure 3F. Next, we found that glucose forms a hydrogen bond network with key amino acids in the substrate recognition pocket of DgpA in figure 3E. Based on this, we designed mutants and evaluated their substrate cleavage activity using kinetic parameters in figure 3J.
Interestingly, during the cleavage of O-glycosidic flavonoids and DgpA/B/C, we observed the formation of intermediate compounds in figure 4A and B.These intermediates were identified as C-glycosidic flavonoids through NMR and LC-MS analysis in figure 4C to H, indicating that DgpA/B/C can convert O-glycosides into C-glycosides.Conclusion. We have pioneered the integration of chemistry and biotechnology to study the structure and functional characteristics of C-glycosides metabolism enzymes, demonstrating that this is a reasonable and practical solution. This approach fills gaps in research methodologies within the field and can be easily applied to other genes with different metabolic functions.
Therefore, it also provides a new reference model for future studies on the structural and functional characteristics of other gut microbial functional proteins.
