勃利霉素对卵菌抑制作用机理研究
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摘要
卵菌包含许多严重危害农作物的病原菌，例如大豆疫霉、马铃薯晚疫病菌等。大豆疫霉引起的大豆疫霉根腐病是一种危害极为严重的世界性大豆病害之一。卵菌病害的防治主要采用化学杀菌剂，甲霜灵(Metalay)作为防治卵菌病害的特效药，目前抗性问题日益严重。因此，研发出更为有效的的生物源杀卵菌剂已是刻不容缓。由链霉菌产生的大环内酯类化合物勃利霉素高效抑制大豆疫霉菌丝生长。勃利霉素对大豆疫霉抑制作用的分子机制还有待深入研究。本研究利用苏氨酸体外添加实验初步确定了勃利霉素对大豆疫霉抑制作用的分子靶标为大豆疫霉的苏氨酰-tRNA合成酶（ThrRS），并通过体外酶活测定、荧光光谱分析和停留光谱等全面分析了勃利霉素对大豆疫霉苏氨酰-tRNA合成酶的抑制作用机理。除此之外，利用生物信息学分析进一步预测了勃利霉素可能对大豆疫霉细胞周期蛋白依赖性蛋白激酶具有抑制作用。结果如下：
（1）在含勃利霉素的大豆疫霉CA培养基中添加苏氨酸，可以明显缓解勃利霉素对大豆疫霉优势小种1菌丝生长的抑制作用，苏氨酸与勃利霉素对大豆疫霉菌丝生长抑制作用之间存在剂量关系，即随着苏氨酸的浓度增加，勃利霉素对大豆疫霉菌丝生长抑制作用减弱，这种现象在其它对照氨基酸中没有观察到，因此可以根据苏氨酸与勃利霉素对大豆疫霉菌丝生长抑制作用之间的剂量效应初步确定勃利霉素对大豆疫霉抑制作用的靶标为苏氨酰-tRNA合成酶。
（2）利用生物信息学分析在大豆疫霉基因组内确定其细胞质苏氨酰-tRNA合成酶为勃利霉素作用靶标。利用PCR基因克隆技术获得大豆疫霉优势小种1的细胞质苏氨酰-tRNA合成酶基因，利用基因工程技术将其克隆到pET32a表达载体，并在大肠杆菌BL21（DE3）中实现了体外表达。利用低温诱导和镍柱纯化获得了可溶性的大豆疫霉细胞质苏氨酰-tRNA合成酶。
（3）建立非放射性方法测定苏氨酰-tRNA合成酶催化苏氨酸活化反应的催化活性：通过离心去除结合到凝胶上的反应生成物ThrRS·Thr~AMP复合物，反应液上清的底物苏氨酸利用氨基酸衍生法和高效液相色谱技术进行检测，由此计算苏氨酸活化反应中生成物的量来测定勃利霉素对大豆疫霉细胞质苏氨酰-tRNA合成酶的酶活性的抑制的IC50。勃利霉素对大豆疫霉细胞质苏氨酰-tRNA合成酶的酶活性的抑制常数为K(app)i为10μM，IC50为8μM，实验测定所用的酶浓度为4μM。由此可见，勃利霉素对大豆疫霉的抑制作用是由抑制大豆疫霉细胞质苏氨酰-tRNA合成酶催化反应引起的。
（4）为进一步明确勃利霉素对大豆疫霉细胞质苏氨酰-tRNA合成酶的抑制作用机制，利用光谱技术研究勃利霉素与大豆疫霉细胞质苏氨酰-tRNA合成酶的相互作用，揭示其结合特性。结果表明勃利霉素对大豆疫霉细胞质苏氨酰-tRNA合成酶的荧光猝灭为静态猝灭，其在大豆疫霉细胞质苏氨酰-tRNA合成酶上只有一个结合位点。两者有较强的结合亲和性，结合常数（bindingconstant） KA为2.552×10~5M~(-1)。热力学常数ΔG <0，ΔH=85.024kJ mol~(-1)，ΔS=0.372kJmol~(-1)K~(-1)，这说明勃利霉素与大豆疫霉细胞质苏氨酰-tRNA合成酶的结合反应是一个自发的过程，主要作用力是疏水作用力。根据F rster非辐射能量转移理论，勃利霉素与大豆疫霉细胞质苏氨酰-tRNA合成酶的结合距离为6.39nm。因此，勃利霉素通过与大豆疫霉细胞质苏氨酰-tRNA合成酶形成复合体发挥其抑制作用。
（5）利用圆二色光谱法研究勃利霉素与大豆疫霉细胞质苏氨酰-tRNA合成酶结合前后，蛋白质微环境及构型的改变。结果表明，勃利霉素与苏氨酰-tRNA合成酶结合导致α-螺旋含量升高，β-折叠含量下降，两者同时发生变化。苏氨酰-tRNA合成酶属于α+β蛋白，由此可知，勃利霉素在与苏氨酰-tRNA合成酶形成复合体的过程中，对其二级结构有较大影响，可能结合在其催化活性位点区域。为进一步确定勃利霉素在大豆疫霉细胞质苏氨酰-tRNA合成酶结合位点与底物结合位点的关系，利用圆二色滴定检测勃利霉素在底物存在下对大豆疫霉细胞质苏氨酰-tRNA合成酶二级结构的影响。结果表明，底物的结合位点与勃利霉素的结合位点并不重叠，勃利霉素是大豆疫霉细胞质苏氨酰-tRNA合成酶非竞争性抑制剂。
（6）利用停留光谱进一步对勃利霉素与疫霉细胞质苏氨酰-tRNA合成酶结合的前稳态动力学特征进行了分析。结果表明，勃利霉素与大豆疫霉苏氨酰-tRNA合成酶快速结合动力学参数kon为4.381×10~6M~(-1)s~(-1)，Kd为22.8nM，勃利霉素与马铃薯晚疫菌苏氨酰-tRNA合成酶kon为4.612×10~6M~(-1)s~(-1)，Kd为21.7nM，接近于勃利霉素对大肠杆菌快速结合动力学参数值(k6on=5×10M-1s-1，Kd<20nM)。底物存在情况下，勃利霉素与大豆疫霉细胞质苏氨酰-tRNA合成酶快速结合仍检测到明显的荧光猝灭作用，进一步说明底物与勃利霉素在大豆疫霉细胞质苏氨酰-tRNA合成酶具有不同的结合位点，勃利霉素为大豆疫霉苏氨酰-tRNA合成酶的非竞争性抑制剂。
（7）多序列比对分析揭示，大豆疫霉细胞质苏氨酰-tRNA合成酶与大肠杆菌苏氨酰-tRNA合成酶具有较高的相似性，大豆疫霉细胞质苏氨酰-tRNA合成酶勃利霉素结合的疏水区(Ser-410,His-412, Cys-437, Pro-438, Leu-593, Phe-597)与预测的大肠杆菌苏氨酰-tRNA合成酶勃利霉素结合区是保守的。利用CASTp-pocket/cavity预测程序预测大豆疫霉细胞质苏氨酰-tRNA合成酶的勃利霉素可能的结合口袋表明，大豆疫霉细胞质苏氨酰-tRNA合成酶与大肠杆菌苏氨酰-tRNA合成酶勃利霉素口袋的氨基酸组成类似，在口袋的体积和面积上有一定差异，大豆疫霉细胞质苏氨酰-tRNA合成酶的勃利霉素结合口袋稍大，可能有利于勃利霉素的结合，提高了大豆疫霉对勃利霉素的敏感性。
（8）作为细胞周期抑制剂，勃利霉素可能对大豆疫霉的细胞周期相关蛋白具有抑制作用。利用序列比对分析、系统进化分析和保守区域分析等预测了与酵母细胞周期蛋白依赖性蛋白激酶Cdc28高度同源的大豆疫霉细胞周期蛋白依赖性蛋白激酶psCdc2，为进一步深入研究勃利霉素对大豆疫霉细胞周期相关蛋白的抑制作用机理奠定了基础。
综上所述，本研究从分子水平上阐明了勃利霉素对大豆疫霉菌丝生长抑制作用的机理，并全面分析了勃利霉素与大豆疫霉细胞质苏氨酰-tRNA合成酶的结合特性、抑制类型和两者结合的对酶构型变化的影响，并对勃利霉素在大豆疫霉细胞质苏氨酰-tRNA合成酶的结合位点进行了预测。我们提出了勃利霉素可能对大豆疫霉细胞周期蛋白有抑制作用，预测了其作用靶标。研究所取得的结果为将勃利霉素开发为新型抗卵菌剂提供了理论基础，对于卵菌病害的防治具有重要意义。
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 constantKA2.552×10~5M~(-1). Thermodynamic analysis by van’t Hoff equation found enthalpy change (ΔH) andentropy change (ΔS) were85.024kJ mol~(-1)and0.372kJ mol~(-1)K~(-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,4.381×10~6M~(-1)s~(-1), and Kd22.8nM, cytoplasm ThrRS of P. infestans with kon,4.612×106M~(-1)s~(-1), and Kd21.7nM, which were similar to E. coli ThrRS(kon=5×10~6M~(-1)s~(-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.
（1）在含勃利霉素的大豆疫霉CA培养基中添加苏氨酸，可以明显缓解勃利霉素对大豆疫霉优势小种1菌丝生长的抑制作用，苏氨酸与勃利霉素对大豆疫霉菌丝生长抑制作用之间存在剂量关系，即随着苏氨酸的浓度增加，勃利霉素对大豆疫霉菌丝生长抑制作用减弱，这种现象在其它对照氨基酸中没有观察到，因此可以根据苏氨酸与勃利霉素对大豆疫霉菌丝生长抑制作用之间的剂量效应初步确定勃利霉素对大豆疫霉抑制作用的靶标为苏氨酰-tRNA合成酶。
（2）利用生物信息学分析在大豆疫霉基因组内确定其细胞质苏氨酰-tRNA合成酶为勃利霉素作用靶标。利用PCR基因克隆技术获得大豆疫霉优势小种1的细胞质苏氨酰-tRNA合成酶基因，利用基因工程技术将其克隆到pET32a表达载体，并在大肠杆菌BL21（DE3）中实现了体外表达。利用低温诱导和镍柱纯化获得了可溶性的大豆疫霉细胞质苏氨酰-tRNA合成酶。
（3）建立非放射性方法测定苏氨酰-tRNA合成酶催化苏氨酸活化反应的催化活性：通过离心去除结合到凝胶上的反应生成物ThrRS·Thr~AMP复合物，反应液上清的底物苏氨酸利用氨基酸衍生法和高效液相色谱技术进行检测，由此计算苏氨酸活化反应中生成物的量来测定勃利霉素对大豆疫霉细胞质苏氨酰-tRNA合成酶的酶活性的抑制的IC50。勃利霉素对大豆疫霉细胞质苏氨酰-tRNA合成酶的酶活性的抑制常数为K(app)i为10μM，IC50为8μM，实验测定所用的酶浓度为4μM。由此可见，勃利霉素对大豆疫霉的抑制作用是由抑制大豆疫霉细胞质苏氨酰-tRNA合成酶催化反应引起的。
（4）为进一步明确勃利霉素对大豆疫霉细胞质苏氨酰-tRNA合成酶的抑制作用机制，利用光谱技术研究勃利霉素与大豆疫霉细胞质苏氨酰-tRNA合成酶的相互作用，揭示其结合特性。结果表明勃利霉素对大豆疫霉细胞质苏氨酰-tRNA合成酶的荧光猝灭为静态猝灭，其在大豆疫霉细胞质苏氨酰-tRNA合成酶上只有一个结合位点。两者有较强的结合亲和性，结合常数（bindingconstant） KA为2.552×10~5M~(-1)。热力学常数ΔG <0，ΔH=85.024kJ mol~(-1)，ΔS=0.372kJmol~(-1)K~(-1)，这说明勃利霉素与大豆疫霉细胞质苏氨酰-tRNA合成酶的结合反应是一个自发的过程，主要作用力是疏水作用力。根据F rster非辐射能量转移理论，勃利霉素与大豆疫霉细胞质苏氨酰-tRNA合成酶的结合距离为6.39nm。因此，勃利霉素通过与大豆疫霉细胞质苏氨酰-tRNA合成酶形成复合体发挥其抑制作用。
（5）利用圆二色光谱法研究勃利霉素与大豆疫霉细胞质苏氨酰-tRNA合成酶结合前后，蛋白质微环境及构型的改变。结果表明，勃利霉素与苏氨酰-tRNA合成酶结合导致α-螺旋含量升高，β-折叠含量下降，两者同时发生变化。苏氨酰-tRNA合成酶属于α+β蛋白，由此可知，勃利霉素在与苏氨酰-tRNA合成酶形成复合体的过程中，对其二级结构有较大影响，可能结合在其催化活性位点区域。为进一步确定勃利霉素在大豆疫霉细胞质苏氨酰-tRNA合成酶结合位点与底物结合位点的关系，利用圆二色滴定检测勃利霉素在底物存在下对大豆疫霉细胞质苏氨酰-tRNA合成酶二级结构的影响。结果表明，底物的结合位点与勃利霉素的结合位点并不重叠，勃利霉素是大豆疫霉细胞质苏氨酰-tRNA合成酶非竞争性抑制剂。
（6）利用停留光谱进一步对勃利霉素与疫霉细胞质苏氨酰-tRNA合成酶结合的前稳态动力学特征进行了分析。结果表明，勃利霉素与大豆疫霉苏氨酰-tRNA合成酶快速结合动力学参数kon为4.381×10~6M~(-1)s~(-1)，Kd为22.8nM，勃利霉素与马铃薯晚疫菌苏氨酰-tRNA合成酶kon为4.612×10~6M~(-1)s~(-1)，Kd为21.7nM，接近于勃利霉素对大肠杆菌快速结合动力学参数值(k6on=5×10M-1s-1，Kd<20nM)。底物存在情况下，勃利霉素与大豆疫霉细胞质苏氨酰-tRNA合成酶快速结合仍检测到明显的荧光猝灭作用，进一步说明底物与勃利霉素在大豆疫霉细胞质苏氨酰-tRNA合成酶具有不同的结合位点，勃利霉素为大豆疫霉苏氨酰-tRNA合成酶的非竞争性抑制剂。
（7）多序列比对分析揭示，大豆疫霉细胞质苏氨酰-tRNA合成酶与大肠杆菌苏氨酰-tRNA合成酶具有较高的相似性，大豆疫霉细胞质苏氨酰-tRNA合成酶勃利霉素结合的疏水区(Ser-410,His-412, Cys-437, Pro-438, Leu-593, Phe-597)与预测的大肠杆菌苏氨酰-tRNA合成酶勃利霉素结合区是保守的。利用CASTp-pocket/cavity预测程序预测大豆疫霉细胞质苏氨酰-tRNA合成酶的勃利霉素可能的结合口袋表明，大豆疫霉细胞质苏氨酰-tRNA合成酶与大肠杆菌苏氨酰-tRNA合成酶勃利霉素口袋的氨基酸组成类似，在口袋的体积和面积上有一定差异，大豆疫霉细胞质苏氨酰-tRNA合成酶的勃利霉素结合口袋稍大，可能有利于勃利霉素的结合，提高了大豆疫霉对勃利霉素的敏感性。
（8）作为细胞周期抑制剂，勃利霉素可能对大豆疫霉的细胞周期相关蛋白具有抑制作用。利用序列比对分析、系统进化分析和保守区域分析等预测了与酵母细胞周期蛋白依赖性蛋白激酶Cdc28高度同源的大豆疫霉细胞周期蛋白依赖性蛋白激酶psCdc2，为进一步深入研究勃利霉素对大豆疫霉细胞周期相关蛋白的抑制作用机理奠定了基础。
综上所述，本研究从分子水平上阐明了勃利霉素对大豆疫霉菌丝生长抑制作用的机理，并全面分析了勃利霉素与大豆疫霉细胞质苏氨酰-tRNA合成酶的结合特性、抑制类型和两者结合的对酶构型变化的影响，并对勃利霉素在大豆疫霉细胞质苏氨酰-tRNA合成酶的结合位点进行了预测。我们提出了勃利霉素可能对大豆疫霉细胞周期蛋白有抑制作用，预测了其作用靶标。研究所取得的结果为将勃利霉素开发为新型抗卵菌剂提供了理论基础，对于卵菌病害的防治具有重要意义。
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 constantKA2.552×10~5M~(-1). Thermodynamic analysis by van’t Hoff equation found enthalpy change (ΔH) andentropy change (ΔS) were85.024kJ mol~(-1)and0.372kJ mol~(-1)K~(-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,4.381×10~6M~(-1)s~(-1), and Kd22.8nM, cytoplasm ThrRS of P. infestans with kon,4.612×106M~(-1)s~(-1), and Kd21.7nM, which were similar to E. coli ThrRS(kon=5×10~6M~(-1)s~(-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.
引文
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