摘要
我国是一个人口多、耕地少的农业大国,农业的增长和可持续发展是我国经济繁荣与社会稳定的基础。农药的使用极大地改善了农业生产,但也面临农药过度使用或单一机制长期大面积使用而产生的抗性问题。如何充分利用已经被实践证明了的超高效除草剂靶标酶,发展新型绿色农药,解决除草剂抗性问题,已经成为全球关注的科学问题。
乙酰羟酸合成酶(acetohydroxyacid synthase, AHAS)是几类商品化超高效除草剂的作用靶标,是催化支链氨基酸生物合成途径中的第一个关键性酶。所有的AHAS都是由催化亚基和调控亚基组成的,催化亚基和调控亚基的相互作用对酶行使全部机制非常重要。我们的研究成果及相关文献表明：AHAS调控亚基激活功能的缺失对AHAS活性的影响程度与除草剂所起的作用相当。并且AHAS亚基间异源重组激活现象说明AHAS亚基间相互作用具有共同的作用界面。这个共同作用界面是潜在的抑制剂靶位,通过模拟相互作用的肽段以及设计靶向相互作用的小分子,可以发展新作用机制的农药活性先导化合物。
迄今为止,AHAS复合物的晶体结构还没有被报道,分子水平上的亚基间相互作用的报道也很少,对于AHAS的催化反应机制、调控亚基对催化亚基的激活机制以及支链氨基酸对全酶活性的反馈调控机制更是知之甚少。因此,AHAS亚基间相互作用的研究对开发新型的农药活性先导化合物具有重要的意义。本论文主要进行了以下几方面的工作：
一、首次建立了蛋白表面扫描标记技术(a global-surface, site-directed labeling scanning method)用于快速鉴定蛋白-蛋白相互作用的关键结构域,并与定点突变技术以及计算机方法相结合提出了一套完整的研究分子水平蛋白-蛋白相互作用的方法。该方法可以广泛的用于研究其他具有相互作用的蛋白。
二、通过以上方法对AHAS亚基间相互作用进行了研究,并确定了调控亚基(ilvH)的Arg26和Asp69是与催化亚基(ilvl)相互作用的关键残基。生物活性数据与pulldown实验结果一致证明：ilvH的Arg26和Asp69是与ilvl相互作用的关键氨基酸残基；Arg26和Asp69侧链的电性对相互作用很重要；并且Arg26和Asp69与ilvI的作用是相互协同的。依次以亚基相互作用的关键区域和关键残基为条件筛选HEX对接得到的AHAS全酶复合物构象,确定了最接近自然状态的复合物结构。依据复合物的结构信息对AHAS的激活和调控机制进行了初步的探讨。
三、催化亚基(ilvI)的六个半胱氨酸全部突变为丙氨酸得到的ilvI'的活性与ilvI相似,可以用ilvI'来代替ilvI进行实验。通过蛋白表面扫描标记的方法初步确定催化亚基Ter308周围可能是与调控亚基相互作用的区域。催化亚基二聚体界面残基的突变研究发现：催化亚基的聚集状态对酶催化活性以及与调控亚基的相互作用有很大的影响。
四. AHAS亚基间的相互作用受反应体系的影响,反应缓冲液pH值对相互作用的影响实验结果说明,极性的环境有利于亚基间的相互作用,暗示了在AHAS亚基相互作用的界面上可能有较多的极性残基。AHAS的催化反应需要金属离子作为辅酶,但是其对金属离子的特异性并不高。同一主族的二价金属离子Mg2+、Ca2+、Sr2+和Ba2+,都可以使大肠杆菌AHAS Ⅲ行使催化功能,但是随着金属离子半径的增加,单独催化亚基和重组酶的活性都在降低,但是调控亚基对催化亚基的激活倍数却在增加。
五、激活大肠杆菌AHAS Ⅲ催化亚基(ilvI)的最小调控亚基(ilvH)的肽段被确定是△N14-△C89,它构成一个完整的ACT结构域,但是不具有反馈调控活性,暗示了调控亚基对催化亚基的激活和反馈调控进行的相互作用是存在差异的。酶活性数据与微量热泳动(MST)实验结果表明：ΔN14-ΔC89不仅可以激活和结合同源的催化亚基ilvI和同种属异源的ilvB(大肠杆菌AHAS I的催化亚基),还可以跨种属激活和结合酵母、拟南芥以及烟草AHAS的催化亚基。而且ΔN14-ΔC89的氨基酸序列在AHAS调控亚基中具有高度的序列相似性。这些实验结果表明在所有的AHAS中存在共同的亚基间相互作用的界面。这为靶向亚基间相互作用开发AHAS抑制剂提供了理论基础。
六、AHAS调控亚基中包含典型的ACT结构域,如同其他的ACT结构域,与效应子(支链氨基酸)结合实现对AHAS催化活性的调控。支链氨基酸对AHAS全酶活性的抑制可以达到80%左右,与磺酰脲类和咪唑啉酮类除草剂的抑制效率相当。通过对二十种编码氨基酸抑制活性的筛选,确定支链氨基酸的抑制能力最强。希望在计算机模拟对接的指导下对支链氨基酸进行化学修饰,获得模仿支链氨基酸反馈抑制机制的新型的农药活性先导化合物。在生物进化过程中,ACT结构域作为一个进化模块,广泛的分布于各种生物体内,出现在各种不同的蛋白中。ACT结构域的氨基酸序列的平均相似性只有16%,但是目前已有的ACT结构域的三维结构却高度相似(RMSD约为2.5A)。AHAS的最小激活肽段构成一个完整的ACT结构域,它的跨种属激活能力引发了我们对ACT结构域调控网络存在的思考。大肠杆菌AHAS Ⅱ的调控亚基(ilvM)与其它AHAS调控亚基的氨基酸序列相似性很低(13-22%),但是ilvM可以使大肠杆菌、酵母、烟草以及拟南芥AHAS催化亚基的活性增加8-16倍,这更坚定了我们对ACT结构域调控网络存在的想法。我们设计构建了三种ACT结构域蛋白,活性数据结果显示：不同的ACT结构域蛋白可以使AHAS的催化亚基活性增加1-2倍,激活所需ACT结构域蛋白的量因AHAS催化亚基的种属不同而存在差异。初步证实了ACT结构域跨途径调控网络存在的可能性。
综上所述,本文首次提出了可快速鉴定蛋白-蛋白相互作用关键区域以及残基的蛋白表面扫描标记技术,与定点突变技术以及计算方法相结合构建了一套完整的研究分子水平蛋白相互作用的方法,该方法可以广泛的用于研究其他具有相互作用的蛋白。本文中通过该方法确定了大肠杆菌AHAS Ⅲ亚基间相互作用的界面以及关键氨基酸残基,建立了AHAS催化亚基与调控亚基相互作用的分子模型。并且对AHAS的催化、激活以及反馈调控机制进行了初步的探讨。研究了反应体系对AHAS亚基间相互作用的影响。确定了激活AHAS催化亚基的最小调控亚基肽段,这个最小激活肽段,构成完整的ACT结构域,具备跨种属异源激活AHAS催化亚基的能力,而且其氨基酸序列在AHAS调控亚基中具有很高的相似性,说明所有的AHAS中存在共同的亚基间相互作用的界面。这为靶向亚基间相互作用开发新作用机制的农药活性先导化合物提供了理论基础。支链氨基酸对AHAS催化活性的抑制程度与商品化的除草剂相当,模仿支链氨基酸的反馈调控,可以用来开发新型作用机制的农药活性先导化合物。在生物进化过程中,ACT结构域作为一个进化模块,出现在各种不同的蛋白中。AHAS最小激活肽段的跨种属激活现象引发了我们对ACT结构域潜在的广谱调控网络存在的思考,通过对ACT结构域的研究初步证实了ACT结构域跨途径调控网络存在的可能性。
China is a large agricultural country with large population and little arable land, hence the growth and sustainable development of agriculture is the foundation of economic prosperity and social stability. The pesticide has greatly improved agricultural production, but also faces the problem of pesticide resistance, duing to overuse or/and single mechanism for long-term use. How to use the proved ultra-efficient target enzymes to develop new green pesticides and solve the resistance problem has became a global scientific issue.
Acetohydroxyacid synthase (AHAS, EC2.2.1.6), which is an important target of action of several commercialized herbicide with high herbicidal efficience, catalyzes the first common step in the biosynthesis of branched chain amino acids (valine, leucine and isoleucine). AHAS is composed of catalytic and regulatory subunits and the enzyme exhibits full activity only when the regulatory subunit (RSU) binds to the catalytic subunit (CSU). Previous results have suggested that the losing of the RSU has the considerable effect on AHAS activation with the herbicides inhibition. Addtionally, the heterologous activation among subunits of AHAS impled that there was a common interface between the CSU and the RSU. Hence, the common interface of AHAS is proposed to be a potential target for developing the new inhibitor with new action mechanism.
The crystal structure of the holoenzyme has not been reported yet, and the molecular interaction between the CSU and the RSU is also unknown. The mechanism of catalytion, activation and feedback inhition was poorly understood. Therefore, the sduty on subunits interaction of AHAS is very important to develop the new pesticide. Work in this thesis was carried out as following:
1. The global-surface, site-directed labeling scanning method is established to efficiently identify the key regions and residues for protein-protein interactions. Combined with the site-directed mutagenesis and computational approach, a protocol was established for the identification of the molecular interactions between proteins.
2. Based on the protocol, the subunits interaction of E. coli AHAS III was studied. The Arg26and Asp69of the regulatory subunit were identified to be the key residues to interact with the catalytic subunit. The results of enzyme activation and pulldown experiment both showed that Arg26and Asp69were the key residues for subunits interaction with cooperative action and the charge of side-chain of Arg26and Asp69played an important role on the subunits interaction. A plausible protein-protein interaction model of the holoenzyme of E. coli AHAS III was proposed, based on the mutagenesis and protein docking studies.
3. The mutant ilvl', which was constructed by substituting all cyscines in the catalytic subunit of E. coli AHAS III (ilvl) with alaines, showed comparable activity with the wild-type ilvl. Hence the ilvl' was able to instead of ilvl in the expriment. The potential interaction region of ilvl was determined to be around the residue Ter308using global-surface, site-directed labeling scanning method. The mutation in the dimer interface of ilvl showed that:the aggregation state of ilvl had effect on the enzymatic activity and interaction with ilvH. Further experimental data on the thermodynamics of subunit associations will be required for further understanding.
4. The nature of reaction buffer has a significant impact on subunit interaction of AHAS. The results of pH dependency experiment showed that the polarity of the environment was conducive to subunits interaction, suggesting that there were more polar residues at the interface. AHAS catalytic reaction requires a divalent metal ion as a coenzyme but it does not have high specificity. The activity of E. coli AHAS Ⅲ can be activated by Mg2+, Ca2+, Sr2+and Ba2+. The enzymatic activity of both the isolated CSV and the reconstituted holoenzyme decreased with increasing the metal ion radius, but the activation ability of the RSU to the CSU increased.
5. The minimum peptide of ilvH to activate ilvl. was determined to be the ΔN14-ΔC89, which comprises a full ACT domain. ΔN14-ΔC89was insensitive to valine inhibition, suggesting different elements for enzymatic activation and feedback regulation. The results of enzymatic activition and microscale thermophoresis (MST) showed that this peptide could not only activate and bind to its homologous ilvI and heterologous ilvB (CSU of E. coli AHAS I), but also heterologously activate and bind to the CSUs of AHAS from Saccharomyces cerevisiae, Arabidopsis thaliana and Nicotiana plumbaginifolia. The high sequence similarity of the peptide ΔN14-ΔC89to RSUs across species hints that this peptide represents the minimum activation motif in RSU and that it regulates all AHASs. These results would shed light on the design of inhibitor or regulator molecular targeted for PPI of AHAS.
6. The RSU of AHAS contains the typical ACT domain, like all the other ACT domains, binding with the effector (BCAA, branched chain amino acids) to regulate the enzymatic activity. The inhibition efficiency of the BCAA was about80%, considerable with sulfonylurea and imidazolinone herbicides. The inhibitory activity of the20encoding amino acids was screened and the BCAA was the most efficient. The inhibition efficiency of the BCAA was considerable with sulfonylurea and imidazolinone herbicides. Therefore, to mimic the feedback regulation of the BCAA could develop the new pesticide active lead compounds with a novel action mechanism. The ACT domain is an evolutionarily mobile ligand binding regulatory module that has been fused to different enzymes at various times. The average sequence similarity of the ACT domian is only16%, but the three-dimensional structure is highly similar (RMSD-2.5A). The AHAS minimum activation peptides comprises a full ACT domain with cross-species activate ability, which triggered the thinking of the existence of the potential and broad spectrum regulatory networks of the ACT domain. The regulatory subunit of E. coli AHAS II (ilvM) has low sequence similarity in the RSU of AHAS (13-22%), but the ilvM can increase the activity of the CSU from E. coli, S. cerevisiae, A. thaliana and N. plumbaginifolia by8-16times. These results confirmed our idea of regulatory networks of the ACT domain. Three ACT domain proteins of different passways had been designed to activate the CSU of AHAS. The activation results suggested that the enzymatic activity of the CSU increased1-2times with the ACT proteins, verifing the presence of a potential and broad spectrum regulatory networks of the ACT domain in different passways.
In summary, this thesis first established a global-surface, site-directed labeling scanning method to rapidly and efficiently identify the key interaction regions and residues for PPI. Combined with the site-directed mutagenesis and computational approach, a protocol was established for determining the molecular interactions of the PPI. Using this protocol, the key residues for subunits interaction of E. coli AHAS III have been indentified and a plausible PPI model of the holoenzyme of E. coli AHAS Ⅲ is proposed. The activation and regulatory mechanism of AHAS has been preliminary studied. The effect of reaction buffer on subunit interaction of AHAS has been discussed. The minimum activation peptide has been determined, which comprises a full ACT domain. This peptide could activate catalytic subunits across the species and has a high sequence similarity with RSUs, suggesting that it represents the minimum activation motif in RSU and regulates all AHASs. The inhibition efficiency of the BCAA was considerable with sulfonylurea and imidazolinone herbicides. Therefore, to mimic the feedback regulation of the BCAA could develop the new pesticide active lead compounds with a novel action mechanism. The ACT domain is an evolutionarily mobile ligand binding regulatory module that has been fused to different enzymes at various times. The AHAS minimum activation peptides comprises a full ACT domain with cross-species activate ability, which triggered the thinking of the existence of the potential and broad spectrum regulatory networks of the ACT domain in different passways.
引文
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