As potential inhibitors target to biological enzymes, antibiotics may have certain impacts on the biochemical treatment process. With micrococcus catalase (CAT) served as the target molecule, the impact and inhibition mechanism for typical tetracyclines (TCs) were evaluated. Toxicity experiments showed that TCs had significant inhibition on CAT in the sequence of tetracycline>chlortetracycline>oxytetracycline>doxycycline. To clarify the inhibition mechanism between TCs and CAT which was explored with the assistance of fluorescence spectroscopy and MOE molecule simulation. According to fluorescence analysis, TCs quenched the fluorescence signal of CAT by the mode of static quenching. Combined with toxicity data, it could be presumed that TCs combined with the catalytic active center and thus inhibited CAT. Above presumption was further verified by the molecular simulation data. When TCs combined with the catalytic center of CAT, the compounds have increased combination areas and prominent energy change (compared with the compounds formed by TCs and noncatalytic center recommend by MOE software). IBM SPSS statistics showed that TC toxicity positively correlated with the hydrogen bonds such as O13→Glu252, O1←Arg195, and O6→Asp249, but negatively correlated with the hydrogen bonds such as O10→Pro363, O10→Lys455, and O12 → Asn127. TC toxicity also positively correlated with the ion bonds ofN4-Glu252, but negatively correlated with the ion bonds of N4-Asp379. Hydrogen bonds and ion bonds for above key sites were closely related to the inhibition effect of TCs on CAT.
TCs have been widely used in animal husbandry and aquaculture as the advantages of low-cost and broad antibacterial spectrum [
In view of TC biotoxicity, controls on their levels become of great importance. At present, the main treatment methods are the chemical method and biochemical method [
Biological enzymes include hydrolases, oxidoreductases, and transferases, which are mainly derived from bacteria, fungi, protozoa, and algae. Catalase (CAT) is an oxidoreductase with iron porphyrin as a prosthetic group [
To clarify the inhibition mechanism, micrococcus catalase was selected to evaluate the impact and inhibition mechanism by typical TCs. The interaction between TCs and CAT was explored with the assistance of fluorescence spectroscopy and molecular docking simulation. According to fluorescence spectroscopy, the impact of TCs on CAT characteristic fluorescence spectra could be obtained to confirm their quenching mechanism. In addition, the inhibition mechanism could be explained by the docking data: combination area, energy change, hydrogen bonds, and ion bonds for key sites. This study offers a comprehensive cognition on TC toxicity regulation and provides valid theoretical support to control their potential risk.
Tetracycline, chlortetracycline, oxytetracycline, and doxycycline were purchased from Meilun Biotechnology Co., Ltd. (Dalian, China). Micrococcus CAT was purchased from Sigma (Saint-Quentin Fallavier, France). Hydrochloric acid, sulfuric acid, sodium hydroxide, H2O2, sodium dihydrogen phosphate, and disodium phosphate were purchased from Sinopharm (Shanghai, China). Methanol and ethanol were obtained from Fisher Chemicals (Fair Lawn, NJ, USA).
Referring to Yang’s ultraviolet spectrophotometry [
Fluorescence spectra were measured by a three-dimensional fluorescence spectrometer (Hiachi Limited, F-7000). Firstly, 2 mL of 0.2 mol/L phosphate buffer (pH 7.8), 1 mL of 400 mg/L CAT solution, and 1 ml different amounts (0 mg/L-1500 mg/L) of TC solution were sequentially added to 50 mL colorimetric tubes. In addition, 2 mL of 0.2 mol/L phosphate buffer (pH 7.8) and 1 mL of 1500 mg/L TC solution were added to another 50 mL colorimetric tube, then make the final volume to 50 ml with ultra-pure water, and mixed and kept for 30 min. The fluorescence spectra of samples were measured at an excitation wavelength of 276 nm and an emission wavelength range of 287-380 nm. Fluorescence quenching type was calculated by the dynamic quenching formula of Stern-Volmer:
Molecular docking simulation was performed with Molecular Operating Environment software (MOE, version 16.09). The main experimental steps are as follows: the model forCAT was obtained from the Protein Data Bank (PDB code 1HBZ,
CAT catalyzes the decomposition of H2O2 into oxygen and water. Therefore, the toxicity of TCs on CAT could be reflected by measuring the absorbance of the remaining H2O2. Figure
Inhibition effect of typical TC target to the CAT activity: (a) tetracycline, (b) chlortetracycline, (c) oxytetracycline, and (d)doxycycline.
Fluorescence spectroscopy can be to study the interaction between proteins and small molecules. When TCs interact with CAT, the fluorescence intensity of CAT will show a dose-effect relationship with TC concentration [
Inhibition effect of different TCs on CAT fluorescence intensity: (a) tetracycline, (b) chlortetracycline, (c) oxytetracycline, and (d) doxycycline.
Stern-Volmer quenching constants for the quenching of TCs on CAT at emission wavelengths of 307.0 nm and 344.4 nm, respectively.
TCs | |||
---|---|---|---|
Tetracycline | 100 mg/L | 2.04 | 2.19 |
300 mg/L | 2.81 | 2.59 | |
500 mg/L | 3.58 | 3.15 | |
1000 mg/L | 4.49 | 4.67 | |
1500 mg/L | 5.45 | 5.05 | |
Chlortetracycline | 100 mg/L | 2.96 | 1.25 |
300 mg/L | 3.43 | 3.22 | |
500 mg/L | 3.00 | 2.51 | |
1000 mg/L | 4.58 | 4.23 | |
1500 mg/L | 5.26 | 4.08 | |
Oxytetracycline | 100 mg/L | 2.27 | 4.41 |
300 mg/L | 3.33 | 4.01 | |
500 mg/L | 3.62 | 4.52 | |
1000 mg/L | 4.44 | 5.46 | |
1500 mg/L | 6.05 | 6.63 | |
Doxycycline | 100 mg/L | 2.07 | 3.16 |
300 mg/L | 5.72 | 6.57 | |
500 mg/L | 3.45 | 4.32 | |
1000 mg/L | 4.85 | 5.81 | |
1500 mg/L | 5.30 | 6.18 |
Based on the toxicity experiment and fluorescence spectroscopy experiment, the interaction for TCs to CAT could be preliminary explained, but the specific molecular mechanism was not clarified. According to molecular docking simulation, the interaction models between TCs and CAT could be obtained, and the discrepant inhibition mechanism could be clarified.
CAT is a binding enzyme with iron porphyrin as a prosthetic group in the catalytic active center. The stereoscopic structure model for CAT was obtained from the Protein Data Bank (PDB code 1HBZ, catalase from micrococcus lysodeikticu
The stereoscopic interaction structure model for TC-CAT complexes (with tetracycline-CAT complexes served as the example).
The interaction between TCs and CAT usually involved in changed combination area and energy. To further clarify the combination site, relevant parameters were obtained by MOE, and the rate of combination areas to small molecule areas and energy changes for TC-CAT complexes at different combinatiolizan sites were calculated (see Table
Combination area ratio and energy change for TC-CAT complexes at different combination sites.
TCs | Combination area ratio for catalytic active center | Combination area ratio for noncatalytic active center | Energy change for catalytic active center (kcal/mol) | Energy change for noncatalytic active center (kcal/mol) | Combination sites |
---|---|---|---|---|---|
Tetracycline | 58% | 53% | |-5.9784| | |-3.2641| | Catalytic active center |
Chlortetracycline | 62% | 57% | |-7.9117| | |-4.8341| | |
Oxytetracycline | 58% | 56% | |-6.9473| | |-4.5725| | |
Doxycycline | 55% | 53% | |-3.5510| | |-3.3425| |
Based on data in the catalytic activity center, the combination area ratio for different TC-CAT complexes was determined as follows: chlortetracycline>tetracycline=oxytetracycline>doxycycline. Besides, the energy change in the catalytic active center for different TCs combined with CAT had different decreased in the sequence of chlortetracycline>oxytetracycline>tetracycline>doxycycline. Combined with toxicity data, combination area ratio and energy change were not well correlated with TC toxicity. The combination area and energy change of chlortetracycline were the largest, which could be related to its special chlorine atom structure. Furthermore, the correlation between TC toxicity and combination area ratio/energy change was evaluated by IBM SPSS statistics (see Figure
The correlation between TC toxicity and combination area ratio/energy change in the catalytic active center.
The specific interaction sites between TCs and CAT can be seen through the ligand interaction diagram provided by the MOE software (see Figure
The general structure and the ligand interaction diagram: (a)the general structure of TCs; (b) The ligand interaction diagram between tetracycline and CAT (with one of them as an example, the order of each atomic name was automatically sorted by software).
Simulation information of the hydrogen bonds for TC-CAT complexes was shown in Figure
Molecular simulation information of the hydrogen bonds for TC-CAT complexes: (a) energy changes of the hydrogen bonds for TC-CAT complexes. (b) The correlation between TC toxicity and hydrogen bonds for main interaction sites: (a)
To clarify the discrepant inhibition effect, the correlation between TC toxicity and hydrogen bonds for main interaction sites was further evaluated by IBM SPSS statistics. Figure
Pearson correlation between TC toxicity and hydrogen bonds for main interaction sites: (a) Asp379, (b) Val367, (c) Asn127, (d) His368, (e) Glu125, (f)Asn369, (g) Arg195, (h) Glu252, and (i) other correlated amino acid residues.
According to statistical analysis, hydrogen bonds related to the key sites of TCs also were ranked (see Figure
Pearson correlation between TC toxicity and hydrogen bonds for TC main interaction sites: (a) O13, (b) O10, (c) N21, (d) 6-ring, (e) O6, (f) O1, (g) O11, (h) O12, and (i) others.
Simulation information for the ion bonds of TC-CAT complexes were shown in Figure
Molecular simulation information of the ion bonds for TC-CAT complexes: (a) energy changes of the ion bonds for TC-CAT complexes; (b) the correlation between TC toxicity and ion bonds.
To clarify the toxicity inhibition mechanism for the discrepant interaction of TCs on CAT, the toxicity of four typical TCs (tetracycline, chlortetracycline, doxycycline, oxytetracycline) on CAT was evaluated. The inhibition sequence was verified as follows: tetracycline>chlortetracycline>oxytetracycline>doxycycline. With the assistance of fluorescence spectroscopy and MOE molecule simulation, the interactions between TCs and CAT were further evaluated. Fluorescence spectroscopy showed that the reduced fluorescence intensity of CAT should be attributed to static quenching between TCs and CAT in the catalytic active center. Molecular simulation showed that combination sites for TC-CAT complexes were in the catalytic active center by calculating the combination area ratio and energy change in the catalytic active center/noncatalytic active center. Combined with toxicity data, the combination area ratio and energy change in the catalytic active center had basically positive correlation with TC toxicity. In addition, combined with the simulation information and related IBM SPSS statistics, TC toxicity was positively correlated with the hydrogen bonds of O13→Glu252, O1←Arg195, and O6→Asp249, but negatively correlated with O10→Pro363, O10→Lys455, and O12→Asn127. By statistical analysis, the interaction between TCs and CAT was closely related to Asp379, Glu252, Arg195, Asn127, Val367, His368, Asn369, Asp249, Lys256, Gln357, Pro363, Lys455, Thr324, and Arg384 residues in CAT. Besides, TC toxicity was positively correlated with the ion bonds of N4-Glu252, but negatively correlated with the ion bonds of N4-Asp379. In conclusion, above interactions could be used as important indexes to evaluate the inhibition mechanism of TCs on the CAT activity. The regulation of TC toxicity could be achieved by weakening or strengthening the hydrogen bonds and ion bonds for some key interaction sites.
The datasets, codes, and corresponding results are available at
The authors declare that there are no conflicts of interest.
Luyao Ren and Qian Wang contributed equally to this work.
This work is supported by National Natural Science Foundation of China (21876103).
Supplementary information.