嗜热类磷酸三酯酶的内酯酶分子进化以提高对有机磷杀虫剂的降解活力
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摘要
自二十世纪五十年代以来，有机磷化合物已被广泛地用于农用杀虫剂。由于有机磷化合物能不可逆抑制动物神经系统中的乙酰胆碱酯酶，破坏神经传递，因此对人及家畜等脊椎动物具有极强的毒副作用。由于杀虫剂的过量和连续使用，许多地区土地、水生态系统被有机磷化合物污染，对人类健康构成巨大威胁。目前去除有机磷污染物的方法(漂白处理、碱水解、焚烧或填埋处理等)反应条件剧烈、副产物具有毒性及腐蚀性，限制了广泛应用。微生物来源的磷酸三酯酶(PTE, EC3.1.8.1)可以广泛水解有机磷杀虫剂，具有环保和原位解毒优势，提供了生物修复新途径。由于磷酸三酯酶均来源于常温微生物，较低的生物学稳定性限制了其实际应用。因此，应用生物技术手段，研发生物学稳定性好、广谱、高效的有机磷杀虫剂降解酶势在必行。通过检索细菌基因组数据库，我们发现嗜热细菌Geobacillus kaustophilus HTA426基因组DNA中含有一个磷酸三酯酶(命名为GkaP)编码序列GK1506(GenBank ID:3183579)，我们对该基因进行了异源表达，并对重组蛋白的酶学性质、催化机制及分子进化等方面开展了系统研究。
重组GkaP具有优异的生物稳定性，它能够有效的水解各种内酯，以及“非专一性”的磷酸三酯酶活力及酯酶活力。因此GkaP应该被归类到近年来新命名的类磷酸三酯酶的内酯酶(posphotriesterase-like lactonase, PLL)家族。酶的催化“非专一性”是趋异进化成新功能的一个显著特征，研究酶的“非专一性”有助于我们理解蛋白的进化及其祖先，因此GkaP成为了研究磷酸三酯酶趋异进化的理想模式酶。为了探索GkaP独特的“非专一性”催化机制，我们对其金属中心附近热点(hot-spot)氨基酸的功能进行了研究，发现Tyr99位点可能与磷酸三酯酶的趋异进化密切相关。其中一个突变体Y99L对乙基对氧磷的催化效率(kcat/Km)提高11倍，但对δ-癸内酯的催化效率降低15倍，使得底物选择性产生了157倍的转化。对Y99L晶体结构分析表明，突变导致了相邻的loop7发生了大约6.6的向外偏移，这种精细的构象变化可能增加了活性中心的柔性，有利于对磷酸三酯底物识别和催化。我们还选择了假定的质子穿梭路径相关的残基(R230和G209)进行了研究。动力学数据表明突变体G209D的磷酸三酯酶和内酯酶活性分别提高了10倍和3倍，该突变体的晶体结构显示一个水分子与R230和D209分别形成了氢键，这导致R230的侧链构象发生偏转最终使得loop7区域背离活性中心发生大约2的移动。我们推测底物结合loop (loop7)的向外偏移可能创造了更大的底物结合空间，使得更有利于酶对不同底物的催化。我们的工作表明通过少数氨基酸取代可能影响底物结合loop区的动态和构象分布，从而改变“非专一性”酶对底物识别和催化的能力。这些结果对于理解“非专一性”酶的催化机制提供了新的线索。
蛋白质工程技术是提高酶的催化效率、改变底物选择性的有效方法。我们以GkaP为模板进化其潜在的有机磷水解能力。结合理性设计和随机突变的策略，经过四轮的突变和筛选(大约10,000个克隆)，获得了若干磷酸三酯酶活力显著提高的突变体。其中最优的突变体26A8C对于有机磷杀虫剂乙基对氧磷(ethyl-paraoxon)的催化效率相比野生型提高了232倍。除此，该突变体对于其它有机磷杀虫剂例如对硫磷(parathion)、地亚农(diazinon)和氯螨硫磷(chlorpyrifos)的活力提高了19-497倍，极大地拓宽了对有机磷杀虫剂的选择性。同时该突变体的内酯酶活力具有766倍的减少，使得GkaP由一个“非专一性”内酯酶转变成了“专一性”的有机磷水解酶。突变体26A8C含有8个氨基酸的取代：F28I、Y99L、T171S、F228L、N269S、V270G、W271C、G273D。通过结构分析发现大部分突变位点(F228L、N269S、V270G、W271C和G273D)均位于底物结合loop7和8上，进一步证明这两段表面loop在磷酸三酯酶乃至整个酰胺水解酶超家族对底物选择性具有重要作用。我们的工作不但有利于揭示磷酸三酯酶的趋异进化机制而且表明GkaP具有极大的进化潜力，它可以通过实验室进化手段创造高效的催化剂从而为有机磷毒物的降解提供潜在的解决方案。
Synthetic organophosphate (OP) compounds have been widely used as agriculturalpesticides since the1950's. More than100OP pesticides are in use worldwide. Continuousand excessive use of OP compounds has led to the contamination of many terrestrial andaquatic ecosystems. OP compounds are highly toxic due to irreversible inhibitacetylcholinesterase (AChE) and disrupt neurotransmission in the central nervous system forall vertebrates. Currently, decontamination of OP compounds utilizes bleach treatment,alkaline hydrolysis, or incineration. In all cases, the conditions are harsh, and the byproductscan be toxic and corrosive. Therefore, enzymatic degradation of OP compound has receivedconsiderable attention since it provides the possibility of both environmentally friendly and insitu detoxification. The phosphotriesterase (PTE, EC3.1.8.1) within the amidohydrolasesuperfamily can hydrolyze a broad range of OP compounds, including most OP pesticides andCWAs. The phosphotriestrerase has been recognized an ideal candidate for OP detoxification.We are interested in developing more robust phosphotriesterase that can be used asbioremediation tools. By searching bacterial genomic database, we found the locus tagGK1506(GenBank ID:3183579) from Geobacillus kaustophilus HTA426encoding aputative phosphotriesterase (GkaP). The GK1506gene was cloned and heterologousexpressed in Escherichia coli. A systematic study was carried out to reseach its enzymaticproperties, catalytic mechanism and molecular evolution.
The recombinant GkaP possessed exceptional biological stability. It was also found toproficiently hydrolyze various lactones, and exhibited promiscuous phosphotriesterase andesterase activities. It shoule be classified into newly emerging phosphotriesterase-lilelactonase (PLL) family. Enzyme promiscuity is a dominant feature for divergent evolution ofnew catalysts. A better understanding of catalytic promiscuity can improve our knowledge ofprotein evolution and ancestry. Therefor, GkaP becomes an ideal model to study enzymepromiscuousty. We investigated the function of hot spots in the active site by site-directedmutagenesis approach. We found that position99in the active site was closely related tosubstrate discrimination. One evolved variant, Y99L, exhibited an11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, butshowed a15-fold decrease toward the lactonase substrate δ-decanolactone, with a157-foldinversion of substrate specificity. Structural analysis of Y99L revealed that the mutationcauses a~6.6outward shift of adjacent loop7, which may increase the flexibility of theactive site and facilitate organophosphate substrate accommodation and catalysis. To furtherexplore the catalytic mechanism, the hypothetical proton shuttle pathway was constructed forresidues Asp266, Arg230, and Gly209. Mutation G209D increased both the phosphotriesteraseand lactonase activities by up to10-and3-fold, respectively. Structural analysis of G209Dvariant identified two simultaneous hydrogen bond bridges between a water molecule andArg230and Asp209, which resulted in a conformational fluctuation of the Arg230side chain anda~2shift of the adjacent loop7. Our results demonstrate that a limited mutation in apromiscuous enzyme may lead to alterations in the dynamics and conformational distributionof substrate-binding loop, which benefit for an alternative binding substrate and efficientcatalysis. These findings provide a new clue for understanding the catalytic mechanism of thepromiscuous enzyme.
Protein engineering techniques are powerful methods to reshaping enzymatic proficiencyand specificity. We selected GkaP as a template to evolve its ancillary OPs hydrolyticcapability. By combining rational and random mutagenesis strategies, we successfullyobtained several active variants after four rounds of mutation and screening (~10,000colonies). Among these, the best variant26A8C demonstrated232-fold improvement overthe wild-type enzyme in reactivity (kcat/Km) for OP pesticide ethyl-paraoxon. This superiorvariant also exhibited high hydrolytic activities over17-497-fold for several targeted OPpesticides, including parathion, diazinon and chlorpyrifos. Concomitantly, the variant26A8Cshowed a767-fold decrease in lactonase activity, producing specialized for OP rather thanlactone hydrolysis. The variant26A8C accumulated eight mutations: F28I, Y99L, T171S,F228L, N269S, V270G, W271C and G273D. The analysis for the mutagenesis sites in theGkaP structure revealed that the key mutations leading to higher phosphotriesterase activityare located in loops7and8(F228L, N269S, V270G, W271C and G273D), In theamidohydrolase superfamily members in particular, loops7and8are most often involved incontacting the ligands in the active site and determining substrate specificity. These results notonly permit us to obtain further insights into the divergence evolution of plosphotriesterasebut also suggested that laboratory evolution of GkaP may lead to potential biological solutionsfor efficient decontamination of neurotoxic OP compounds.
重组GkaP具有优异的生物稳定性，它能够有效的水解各种内酯，以及“非专一性”的磷酸三酯酶活力及酯酶活力。因此GkaP应该被归类到近年来新命名的类磷酸三酯酶的内酯酶(posphotriesterase-like lactonase, PLL)家族。酶的催化“非专一性”是趋异进化成新功能的一个显著特征，研究酶的“非专一性”有助于我们理解蛋白的进化及其祖先，因此GkaP成为了研究磷酸三酯酶趋异进化的理想模式酶。为了探索GkaP独特的“非专一性”催化机制，我们对其金属中心附近热点(hot-spot)氨基酸的功能进行了研究，发现Tyr99位点可能与磷酸三酯酶的趋异进化密切相关。其中一个突变体Y99L对乙基对氧磷的催化效率(kcat/Km)提高11倍，但对δ-癸内酯的催化效率降低15倍，使得底物选择性产生了157倍的转化。对Y99L晶体结构分析表明，突变导致了相邻的loop7发生了大约6.6的向外偏移，这种精细的构象变化可能增加了活性中心的柔性，有利于对磷酸三酯底物识别和催化。我们还选择了假定的质子穿梭路径相关的残基(R230和G209)进行了研究。动力学数据表明突变体G209D的磷酸三酯酶和内酯酶活性分别提高了10倍和3倍，该突变体的晶体结构显示一个水分子与R230和D209分别形成了氢键，这导致R230的侧链构象发生偏转最终使得loop7区域背离活性中心发生大约2的移动。我们推测底物结合loop (loop7)的向外偏移可能创造了更大的底物结合空间，使得更有利于酶对不同底物的催化。我们的工作表明通过少数氨基酸取代可能影响底物结合loop区的动态和构象分布，从而改变“非专一性”酶对底物识别和催化的能力。这些结果对于理解“非专一性”酶的催化机制提供了新的线索。
蛋白质工程技术是提高酶的催化效率、改变底物选择性的有效方法。我们以GkaP为模板进化其潜在的有机磷水解能力。结合理性设计和随机突变的策略，经过四轮的突变和筛选(大约10,000个克隆)，获得了若干磷酸三酯酶活力显著提高的突变体。其中最优的突变体26A8C对于有机磷杀虫剂乙基对氧磷(ethyl-paraoxon)的催化效率相比野生型提高了232倍。除此，该突变体对于其它有机磷杀虫剂例如对硫磷(parathion)、地亚农(diazinon)和氯螨硫磷(chlorpyrifos)的活力提高了19-497倍，极大地拓宽了对有机磷杀虫剂的选择性。同时该突变体的内酯酶活力具有766倍的减少，使得GkaP由一个“非专一性”内酯酶转变成了“专一性”的有机磷水解酶。突变体26A8C含有8个氨基酸的取代：F28I、Y99L、T171S、F228L、N269S、V270G、W271C、G273D。通过结构分析发现大部分突变位点(F228L、N269S、V270G、W271C和G273D)均位于底物结合loop7和8上，进一步证明这两段表面loop在磷酸三酯酶乃至整个酰胺水解酶超家族对底物选择性具有重要作用。我们的工作不但有利于揭示磷酸三酯酶的趋异进化机制而且表明GkaP具有极大的进化潜力，它可以通过实验室进化手段创造高效的催化剂从而为有机磷毒物的降解提供潜在的解决方案。
Synthetic organophosphate (OP) compounds have been widely used as agriculturalpesticides since the1950's. More than100OP pesticides are in use worldwide. Continuousand excessive use of OP compounds has led to the contamination of many terrestrial andaquatic ecosystems. OP compounds are highly toxic due to irreversible inhibitacetylcholinesterase (AChE) and disrupt neurotransmission in the central nervous system forall vertebrates. Currently, decontamination of OP compounds utilizes bleach treatment,alkaline hydrolysis, or incineration. In all cases, the conditions are harsh, and the byproductscan be toxic and corrosive. Therefore, enzymatic degradation of OP compound has receivedconsiderable attention since it provides the possibility of both environmentally friendly and insitu detoxification. The phosphotriesterase (PTE, EC3.1.8.1) within the amidohydrolasesuperfamily can hydrolyze a broad range of OP compounds, including most OP pesticides andCWAs. The phosphotriestrerase has been recognized an ideal candidate for OP detoxification.We are interested in developing more robust phosphotriesterase that can be used asbioremediation tools. By searching bacterial genomic database, we found the locus tagGK1506(GenBank ID:3183579) from Geobacillus kaustophilus HTA426encoding aputative phosphotriesterase (GkaP). The GK1506gene was cloned and heterologousexpressed in Escherichia coli. A systematic study was carried out to reseach its enzymaticproperties, catalytic mechanism and molecular evolution.
The recombinant GkaP possessed exceptional biological stability. It was also found toproficiently hydrolyze various lactones, and exhibited promiscuous phosphotriesterase andesterase activities. It shoule be classified into newly emerging phosphotriesterase-lilelactonase (PLL) family. Enzyme promiscuity is a dominant feature for divergent evolution ofnew catalysts. A better understanding of catalytic promiscuity can improve our knowledge ofprotein evolution and ancestry. Therefor, GkaP becomes an ideal model to study enzymepromiscuousty. We investigated the function of hot spots in the active site by site-directedmutagenesis approach. We found that position99in the active site was closely related tosubstrate discrimination. One evolved variant, Y99L, exhibited an11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, butshowed a15-fold decrease toward the lactonase substrate δ-decanolactone, with a157-foldinversion of substrate specificity. Structural analysis of Y99L revealed that the mutationcauses a~6.6outward shift of adjacent loop7, which may increase the flexibility of theactive site and facilitate organophosphate substrate accommodation and catalysis. To furtherexplore the catalytic mechanism, the hypothetical proton shuttle pathway was constructed forresidues Asp266, Arg230, and Gly209. Mutation G209D increased both the phosphotriesteraseand lactonase activities by up to10-and3-fold, respectively. Structural analysis of G209Dvariant identified two simultaneous hydrogen bond bridges between a water molecule andArg230and Asp209, which resulted in a conformational fluctuation of the Arg230side chain anda~2shift of the adjacent loop7. Our results demonstrate that a limited mutation in apromiscuous enzyme may lead to alterations in the dynamics and conformational distributionof substrate-binding loop, which benefit for an alternative binding substrate and efficientcatalysis. These findings provide a new clue for understanding the catalytic mechanism of thepromiscuous enzyme.
Protein engineering techniques are powerful methods to reshaping enzymatic proficiencyand specificity. We selected GkaP as a template to evolve its ancillary OPs hydrolyticcapability. By combining rational and random mutagenesis strategies, we successfullyobtained several active variants after four rounds of mutation and screening (~10,000colonies). Among these, the best variant26A8C demonstrated232-fold improvement overthe wild-type enzyme in reactivity (kcat/Km) for OP pesticide ethyl-paraoxon. This superiorvariant also exhibited high hydrolytic activities over17-497-fold for several targeted OPpesticides, including parathion, diazinon and chlorpyrifos. Concomitantly, the variant26A8Cshowed a767-fold decrease in lactonase activity, producing specialized for OP rather thanlactone hydrolysis. The variant26A8C accumulated eight mutations: F28I, Y99L, T171S,F228L, N269S, V270G, W271C and G273D. The analysis for the mutagenesis sites in theGkaP structure revealed that the key mutations leading to higher phosphotriesterase activityare located in loops7and8(F228L, N269S, V270G, W271C and G273D), In theamidohydrolase superfamily members in particular, loops7and8are most often involved incontacting the ligands in the active site and determining substrate specificity. These results notonly permit us to obtain further insights into the divergence evolution of plosphotriesterasebut also suggested that laboratory evolution of GkaP may lead to potential biological solutionsfor efficient decontamination of neurotoxic OP compounds.
引文
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