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活性中心嵌入loop肽段策略创制新功能酶
中文摘要

有机磷化合物(包括有机磷杀虫剂和化学战神经毒剂)能不可逆抑制脊椎动物体内神经系统的乙酰胆碱酯酶,其施用后的残留对人及家畜等产生的剧毒作用已引起高度关注。微生物来源的磷酸三酯酶(EC3.1.8.1,Phosphotriesterases,PTE)具有降解有机磷化合物的活力,提供了环境生物修复新途径。然而常温微生物来源的磷酸三酯酶由于受自然进化的限制,通常稳定性不高,局限了这类酶的实际应用。在对酶结构-功能关系了解的基础上,探索酶趋异进化规律,构建进化高催化活性及高稳定性的新酶,将有望显著提升酶实际应用价值。因为酶活性中心提供了最优的微环境以促进催化反应的进行,改变酶活性中心内的相关残基可对催化过程产生很大影响,甚至可能是酶分子获得新的催化活性的进化途径,因此发展新型蛋白质工程技术,从稳定性高的嗜热微生物酶出发,利用同一超家族酶所具有的相似结构和催化机制,对酶活性中心进行重塑,不仅将促进酶生物技术的实践发展,创制具有PTE催化功能且高稳定性的新酶,而且也将在理论上探索酶分子进化的途径。本论文选定嗜热微生物Geobacillus kaustophilus中的具有微弱磷酸三酯酶活力的热稳定内酯酶(posphotriesterase-like lactonase,GkaP-PLL)为分子设计改造目标,开展了以下研究工作: 1.热稳定性GkaP-PLL内酯酶向PTE功能转化的分子设计:内酯酶PLL和磷酸三酯酶PTE都具有(α/β)₈桶状结构,属于酰胺水解酶超家族,该家族成员的共同显著特征是活性中心位点具有单核或双核金属中心,能够增强底物分子解离基团键的断裂,极化水分子增强亲核进攻。结构分析可发现GkaP-PLL与磷酸三酯酶及其他内酯酶家族成员具有相似的空间结构,值得注意的是GkaP-PLL与高活力的磷酸三酯酶pdPTE金属中心几近吻合, RMSD为0.36Å,两个酶较为显著差异仅在酶活性中心上方表而loop的长度上,而GkaP-PLL相比pdPTE的表面loop7要少11个残基。事实上已报道的PTE被认为相对PLL在活性中心loop7包含着2个独立的插入结构区,L7-A和L7-B,被4个保守氨基酸分隔。高活性的pdPTE相较GkaP-PLL的loop7在L7-A和L7-B区分别存在着2个和9个额外的残基。活性中心loop的构象变化经常也参与许多酶的反应通道的调节和底物分子的结合,因此loop改变可能是酶功能进化的一个基础。对GkaP-PLL酶的关键loop7区域进行了分子设计,即拓展其长度及构成,探索重塑该酶活性中心,有望获得更高PTE催化活性且高稳定性的新酶。迄今为止,由于蛋白折叠的复杂性,通过活性位点loops插入/删除来改造酶活性的方法和研究案例依然稀缺。本研究中,我们设计了活性中心逐步嵌入loop肽段策略(Stepwise Loop Insertion Strategy,StLois),即构建小型smart突变库中筛选获得最优突变体:并将此作为下一轮模版,继续迭代引入突变残基,该方法可以通过有效地校对活性位点loop的突变适应度,来优化突变酶的底物特异性。 2.应用StLois策略建立系列小型智能突变文库和筛选:研究中我们使用前期构建的野生型GkaP-PLL及ML7(26A8F281/Y99L/T171S/F228L/N269S/V270G/G273D)(磷酸三酯酶活性比野生型高38倍的GkaP-PLL定点突变体)重组表达质粒作为模板,进行loop7的肽段嵌入重构。利用带有简并密码子的引物,通过模板的全质粒PCR一次将20种可能的天然氨基酸全部随机引入GkaP-PLL的编码序列,实现引入肽段的饱和突变后,转化E coli BL21-CodonPlus(DE3)-RIL进行筛选。基于算法Pi=1-(1-Fi)〓,为保证编码20种氨基酸的32种密码子在库中被筛选到的机率大于95%,我们构建的小型随机饱和突变库中loop引入肽段的“突变步长”设定在2、3、4残基,筛选的库容量必须分别达到3.0×10³, 9.8×10⁴和3.1×10⁶克隆数。每轮引入的“突变步长”决定着突变库的筛选的效率和有效性,因此是逐步重构酶活性中心loop的关键考虑因素。为了避免大量的耗时的库筛选,提高酶工程效率,我们选定了突变步长设定在2残基随机饱和突变引入肽段,即采用每次随机引入两个突变残基,多轮筛选小型smart突变库积累优良突变效果。 3.突变体性质表征:研究中获得的在L7-A区有着双插入的ML7-A2(DN)突变体有着116倍PET活性增强相对野生型,而L7-B区有着双插入的ML7-B2(VN)突变体有着261倍PET活性增强相对野生型,即6.9倍活性增加对比模板ML7突变体。考虑的ML7-B2(VN)有着更好的有机磷水解活性,其被选作模板进一步对loop7进行残基对的嵌入拓展。基于StLois策略,经过多轮的突变肽段嵌入拓展内酯酶GkaP-PLL活性位点loop7,筛选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GkaP-PLL突变株ML7-B6(VNLGKY)对底物乙基对氧磷的催化效率进一步提高了16倍,催化效率为k〓/K〓=7.9×10⁴M⁻¹s⁻¹,相对于野生型内酯酶,PTE酶活提高达609倍。同时该突变体的内酯酶活力显示了10³到10⁴倍减少,产生了超过10⁷倍的底物特异性转变,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。GkaP-PLL突变株ML7-B6在65℃催化效率为k〓/K〓2.4×10⁵M⁻¹s⁻¹,比先前报道的SsoPox-PLL和DrPLL工程酶活性高了10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17-1252倍,极大地拓宽了对有机磷杀虫剂的选择性。 4.野生型和loop延伸突变体的结构分析:为了分析新的有机磷水解活性的机理,我们试图解析的几种loop延伸突变酶的晶体结构。然而,只有四个残基肽段嵌入变体L7-A2B2(DR-MI)成功结晶被解析,最终分辨率在1.9Å(PDB代码:5CH9)。该loop延伸4个残基突变酶的晶体结构显示突变中的α金属离子和loop7残基V239之间的距离由对应的野生型中10.5Å距离改变为15.4Å。此外,loop3部分肽段(从98位到105位残基)在突变酶L7-A282有着明显向外偏移,导致loop2的D73残基到loop3的A105残基间距离由2.7Å变为了9.4Å,这使活性位点的主入口的宽度明显扩展。我们推测延伸loop7可直接导致活性中心关键催化loops的位移和构象变化,重塑了酶活性中心的催化微环境,相对野生型内酯酶创造了更大的底物结合空间和底物入口。通过分子对接和动力学模拟研究,进一步揭示该突变酶形成了比野生型形成了更有利于有机磷酸酯底物结合的活性位点口袋。 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。这项工作首次建立高效的活性中心loop嵌入策略(StLois),显示了loop重塑在快速产生新型酶功能的重要作用。 关键词:loop插入突变,酶活性位点,底物特异性,蛋白质工程,内酯酶,磷酸三酯酶;

英文摘要

Synthetic organophosphate compounds and their derivatives are highly toxic due to irreversible inhibit acetyl cholinesterase (AChE) and disrupt neurotransmission in the central nervous system for all vertebrates. The phosphotriesterase (PTE, EC3.1.8.1) within the amidohydrolase superfamily can hydrolyze a broad range of OP compounds, including most OP pesticides and chemical warfare agents (CWAs). The phosphotriestrerase (PTE) has been recognized an ideal candidate for OP detoxification but it is less stable and will be inactivated at high temperature. Due to the active site provides an optimal microenvironment for specific catalytic reactions, modification on relevant residues around the active center of enzyme would have a direct influence on catalytic properties of enzymes, which might provide an evolutionary pathway for creating new catalytic activities of enzyme. In this study, thermophilic lactonease (GkaP-PLL) with weak phosphotriesterase activity in Geobacillus kaustophilus was selected as a model for molecular design. To evolve and obtain a better PTE enzyme, we have performed the following research work: 1.Molecular designing to transform a Thermostable Gka/P-PLL lactonase into a PIE enzyme. The PTE and a PTE-like lactonase (PLL) belong to the amidohydrolase superfamily, sharing a common (α/β)₈ TIM-barrel structural fold. GkaP-PLL exhibits a similar folded structure as pdPTE but possesses different active loop configurations. Notably, loop7 of GkaP-PLL is 11 amino acids shorter than that of pdPTE. The PTE loop7 has been proposed to contain two possible individual insertion sites, annotated L7-A and L7-B, connected by a 4-amino-acid spacer. There are two more residues in pdPTE L7-A and nine more in L7-Bthan there are in GkaP-PLL loop7. In addition, there are significant differences in the amino acid constitution and spatial architecture. To efficiently elongate GkaP-PLL loop7, we developed stepwise loop insertion strategy (StLois) strategy to design and construct smart mutant libraries that introduced two residues in a stepwise manner with degenerate NNK codon randomization. The residues were gradually introduced into the Gka/P-PLL L7-A region by saturation mutagenesis. This method can effectively accumulate the active site loop mutation adaptability, to optimize the mutant enzyme of the substrate specificity. 2.Use StLois strategy to construct a series of small intelligent mutant libraries and screening PTE activity. To obtain a more efficient OP hydrolase, we used wild-type GkaP-PLL and our GkaP-PLL variant ML7 (α/β)₈, F28I/Y99L/T171S/F228L/N269S/V270G/G273D), which has approximately 38 times higher activity than the WT, as templates for loop7 insertion mutagenesis. To avoid a massive, time-consuming library screen, we propose that a saturated mutagenesis library of double residue insertions in the loop would be suitable for each round and that the mutational fitness effect could be compounded to identify stable variants with desired functions. The algorithm P〓=l-(1-F〓)〓 was used to count the number of transformants for double (3.0×10³), triple (9.8×10⁴)and quadruple (3.1×10⁶)residue insertions/deletions/mutations as a function of NNK codon degeneracy with 95% coverage. 3.Characterization of evolved variants By inserting six residues into active site loop 7, the best variant ML7-B6 demonstrated a 16fold further increase in catalytic efficiency (K〓/K〓=7.9×10⁴ M⁻¹s⁻¹) toward ethyl-paraoxon compared with its initial template ML7 and 609-fold higher than wild type. The best variant shifts substrate specificity >10⁷ -fold. The catalytic efficiency of OP hydrolysis has been determined for all of the variants at 37℃. In addition, all of the obtained variants exhibited significantly higher hydrolytic activities for the tested pesticides (from 17- to 1252-fold compared to the WT). All of the variants showed a higher activity for ethyl-substituted OP derivatives, including ethyl-paraoxon, ethyl parathion, and diazinon, with the exception of the OP chlorpyrifos. 4.Structural and molecular dynamic analysis of wild-type and loop-extended enzyme variants To address the mechanism of OP hydrolysis with the novel catalysts, we attempted to solve the crystal structures of several variants for structural analysis. However, only variant L7-A2B2 (DR-MI insertion) evolved from wild type template, was successfully crystallized and determined at a resolution of 1.9 Å(PDB code: 5CH9). The variant adopts a fold similar to that of the WT, but striking difference a used by the amino acid insertion in loop7 is evident in the active site pocket. The width of the main entrance to the active site, as measured from the D73 of loop2 to A105 of loop3, was 9.4 Å in the L7-A2B2 variant but only 2.7 Å in the WT, indicating an enlargement of the active site entrance caused by the additional loop residues. The pocket volume for substrate binding in L7-A2B2 is 1124 ų as measured by CASTp online, which is larger than that in WT (430 ų) and might be more suitable for larger OP substrate catalysis. We also performed 80 ns MD simulations on WT and L7-A2B2 mutant complexes. The binding free energy of L7-A2B2 was -28.5±1.3 kcal/mol, which is 42.3% lower than that of the WT (-20.1±0.8 kcal/mol). The RMSD and RMSF analysis suggest that the mutant L7-A2B2 is more stable with ethyl paraoxon compared to δ-decanolactone. Generally, this study demonstrates that StLois is a powerful method for active site remodeling to alter enzyme substrate preference and create new catalysts. The successful change in enzyme function without disrupting the original protein structure has deepened our understanding of the factors that govern molecular evolution in nature. Moreover, this methodology can be readily generalized to design and create novel biocatalysts and even novel functional proteins to perform a wide range of chemical or physiological reactions for which natural enzymes/proteins do not exist. Keywords: loop insertion, active site, substrate specificity, protein engineering, lactonase, phosphotriesterase;

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