Research On The Mechanism Of Antifungal Activity Of Borrelidin Against Oomycete

Oomycete includes many destructive pathogens of plants, such as Phytophthora sojae Kaufm.&Gerd., Phytophthora infestans （Mont.） de Bary and so on. Phytophthora root rot, caused by P. sojae, isone of the most devastating diseases of soybean（Glycine max （L.） Merr.） in the world. The main meansof the control of oomycete disease was chemical fungicide, and metalay was used widespread, butresistance to fungicides in plant pathogens had become an outstanding problem in chemical control ofoomycete diseases. So, it is urgent need to developing a novel agricultural antifungal agent againstoomycete. Macrolide antibiotics borrelidin produced by streptomycete has high and specific anti-fungalactivity against P. sojae. Molecular mechanism of antifungal of borrelidin against P. sojae need furtherresearch. This research primarily revealed cytoplasm threonyl-tRNA synthetase （ThrRS） of P. sojae astarget of borrelidin by threonine addition experiment, and the antifungal activity of borrelidin against P.sojae mediated by inhibition of ThrRS was analyzed by measure of enzyme activity, fluorescencespectroscopy, circular dichroism （CD） and stopped-flow transient kinetic analysis. Beyond that, wepredicted cycle-dependent kinase of P. sojae by bioinformatics method, which is probable to beinhibited by borrelidin. The results were as follows:（1） The growth inhibition of P. sojae race1caused by borrelidin could be attenuated by threoninein borrelidin containing CA medium, and there is a dose-dependent manner between threonine andantifungal activity of borrelidin against P. sojae, namely, antifungal activity of borrelidin against P.sojae became weak with the increasing of the concentration of threonine, and the attenuation in adose-dependent manner was specific for threonine, and other amino acids as control did not influencethe inhibitory effect of borrelidin. These results clearly indicated the involvement of ThrRS inantifungal activity of borrelidin against P. sojae, and ThrRS may be the potential target of borrelidin.（2） The cytoplasm ThrRS of P. sojae was indentified in the genome of P. sojae by bioinformaticsmethod as the target of borrelidin. The cytoplasm ThrRS of P. sojae（psTRS） was gained by PCRamplication method, and was cloned into the pET32a expression vector using gene engineeringtechnology, then the recombinant pET32a expression vector was transformed into Escherichia coliBL21（DE3） which were induced at the low temperature, and P. sojae ThrRS protein was purified bynickel-nitrilotriacetic acid-agarose chromatography.（3） A non-radioactive method was established to detect the activity of ThrRS in the catalyticreaction of threonine activation: After the reaction products ThrRS·Thr^AMP complex binding toagarose were removed by centrifugation, substrate threonine in the supernatant of the reaction liquidwas determined by the amino acid derivative method and the high-performance liquid chromatographytechnique（HPLC）, and the amount of the reaction products was calculated to measure IC50of theinhibition of the enzymatic activity of ThrRS by borrelidin in vitro.The results showed that borrelidin can significantly inhibit the cytoplasm ThrRS（4μM） enzymatic activity with a K（app）iand IC50value of10μM and8μM, respectively, confirming the assumption that the growth inhibition of P. sojae wascaused by the decreased enzymatic activity of cytoplasm ThrRS associated with borrelidin.（4） In order to confirm the inhibition effect of borrelidin on cytoplasm ThrRS of P. sojae andinvestigate the interaction between them, fluorescence spectroscopy was employed to study the bindingproperty of borrelidin to cytoplasm ThrRS of P. sojae. The results showed that borrelidin caused a staticquenching of intrinsic fluorescence of cytoplasm ThrRS, and there was one primary borrelidin bindingsite on ThrRS. Borrelidin has a strong binding affinity for cytoplasm ThrRS with the binding constantKA2.552×10⁵M^-1. Thermodynamic analysis by van’t Hoff equation found enthalpy change （ΔH） andentropy change （ΔS） were85.024kJ mol^-1and0.372kJ mol^-1K^-1, respectively, which indicated that thehydrophobic forces played important roles in the binding of borrelidin and cytoplasm ThrRS. Thedistance r=6.39nm between donor （ThrRS） and acceptor （borrelidin） was estimated based on theF rster theory of non-radiative energy transfer. So, the antifungal activity of borrelidin against P. sojaewas mediated by inhibition of cytoplasm ThrRS via the formation of ThrRS-borrelidin complex.（5） The change of the microenvironment and conformation of cytoplasm ThrRS in the bindingreaction was studied by circular dichroism （CD）. The results demonstrated that the binding of borrelidincould induce the conformational changes of cytoplasm ThrRS with increased content of α-helicity anddecreased content of β-sheet, and α-helicity and β-sheet of cytoplasm ThrRS both changed after theformation of complex of ThrRS-borrelidin. Because the folding of ThrRS is an alpha and beta protein （α+β）, the binding of borrelidin had a great impact on the secondary structure of cytoplasm ThrRS, andwe proposed that borrelidin binds in the catalytic region of cytoplasm ThrRS. To further determine therelationship between borrelidin binding site and substrate binding sites on cytoplasm ThrRS of P. sojae,the CD titration was used to investigate the effect of borrelidin binding on secondary structure of ThrRSin the present of substrate. The results indicated that borrelidin has not overlapping binding sites withthe enzyme substrates, and borrelidin is a noncompetitive inhibitor of cytoplasm ThrRS of P. sojae withrespect to threonine and ATP.（6） Presteady-state kinetic analysis of the binding process of borrelidin and ThrRS was carried outby stopped-flow. The stopped-flow results showed that borrelidin binds to cytoplasm ThrRS of P. sojaewith kon,4.381×10⁶M^-1s^-1, and Kd22.8nM, cytoplasm ThrRS of P. infestans with kon,4.612×106M^-1s^-1, and Kd21.7nM, which were similar to E. coli ThrRS(kon=5×10⁶M^-1s^-1，Kd<20nM). In the presentof substrate, the enzyme fluorescence is still quenched by borrelidin, which indicated that borrelidin hasa different binding site from subatrate binding site, as expected for a noncompetitive inhibitor of P.sojae ThrRS.（7） Multiple sequence alignment analysis revealed P. sojae ThrRS and E. coli ThrRS had highsimilarity, the hydrophobic cluster of cytoplasm ThrRS of P. sojae （Ser-410, His-412, Cys-437, Pro-438,Leu-593, Phe-597） is conserved with that of E. coli ThrRS. The binding pocket of borrelidin incytoplasm ThrRS of P. sojae predicted by CASTp-pocket/cavity program showed that amino acidcomposition of the binding pocket of borrelidin in cytoplasm ThrRS of P. sojae and E. coli ThrRS wassimilar, and there are some differences in the volume and area of the pocket. The binding pocket ofborrelidin in cytoplasm ThrRS of P. sojae was some bigger than that of E. coli ThrRS, which may bebeneficial to improve the sensitivity of P. sojae to borrelidin. （8） As inhibitor of cell cycle, we proposed that borrelidin inhibit the cell cycle relative protein. Thecycle-dependent kinase of P. sojae psCdc2was identified to homology with the cycle-dependent kinaseof E. coli Cdc28by BLAST, phylogenetic analysis and conserved regions search, which provided thebasis for further study of inhibition of borrelidin to cycle-dependent kinase.In summary, These studies provide insight into the molecular mechanism of antifungal activity ofborrelidin against P. sojae, and the interaction of borrelidin and cytoplasm ThrRS, inhibitor type andconformational changes of cytoplasm ThrRS was comprehensively analyzed, meanwhile, the borrelidinbinding site and pocket in cytoplasm ThrRS of P. sojae were predicted. Moreover, we proposed thatborrelidin may inhibit cell cycle relative protein of P. sojae, the target gene was predicted. The resultsobtained here provide a theoretical foundation for the development of borrelidin as a novelanti-oomycete agent, and was a great significance for the prevention and control of oomyce diseases.