CN111172130B - 一组拟南芥磷酸核酮糖激酶的突变体 - Google Patents
一组拟南芥磷酸核酮糖激酶的突变体 Download PDFInfo
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Abstract
本发明涉及一组拟南芥磷酸核酮糖激酶的突变体,所述的突变体为:在如SEQIDNO.1所示的野生型AtPRK蛋白的序列基础上,突变了如下位点形成的突变体:(1)D14A、S15A、K19A、S20A、W156A中的任一或任意组合;(2)D58A、H61A、R65A、R68A、K69A、Y104F、H106A中的任一或任意组合;或(3)上述两组突变位点的任意组合;其中第(1)组突变涉及ATP亲和力的变化,第(2)组突变涉及Ru5P亲和力的变化。本发明还涉及所述的突变体的应用,(1)构建改变卡尔文循环(暗反应阶段)功能的模型植株;(2)构建植物模型;(3)构建具有改进的固碳功能的植株;(4)催化5‑磷酸核酮糖(Ru5P)为1,5‑二磷酸核酮糖。
Description
技术领域
本发明属于生物技术领域,具体而言,涉及一组拟南芥磷酸核酮糖激酶的突变体。
背景技术
光合作用是地球上最为重要的物理化学反应之一,光合生物利用光能将二氧化碳和水转换为碳水化合物。放氧光合生物,如蓝藻,绿藻和高等植物等还能释放出氧气。光合作用分为光反应阶段和暗反应阶段,其中光反应是严格依赖于光的,而暗反应不需要光也能进行。在光反应阶段中,光系统吸收光能,在反应中心裂解水释放出氧气、质子和电子,同时生成烟酰胺腺嘌呤二核苷酸磷酸(NADPH)和三磷酸腺苷(ATP)。在暗反应阶段,光合生物利用光反应阶段产生的NADPH和ATP,固定二氧化碳,合成糖类,这是一个由多个酶参与的多步催化反应过程,被称为卡尔文循环。虽然卡尔文循环不依赖光,但是需要光反应过程中生成的ATP和NADPH,所以卡尔文循环的进行也受到“光照/黑暗”转换的调控,这一过程主要通过叶绿体类囊体基质中的氧化还原状态变换来调节的。参与卡尔文循环的11个酶中有4个是受到氧化还原调控的,还原态是它们的活性形式,氧化态则是失活状态。在光照条件下,光系统I(PSI)激活“铁氧还蛋白-硫氧还蛋白”氧化还原系统,还原叶绿体中的硫氧还蛋白(TRX)。还原态的硫氧还蛋白可以继续还原并激活卡尔文循环中的酶,其中磷酸核酮糖激酶(PRK)和甘油醛-3-磷酸脱氢酶(GAPDH)都受其调控。此外,叶绿体中的一个小蛋白叶绿体蛋白12(chloroplast protein 12,CP12),作为卡尔文循环调控的中心,也受到硫氧还蛋白的调控。
暗反应阶段通常被分为三个过程:二氧化碳固定,三碳糖还原和五碳糖再生。PRK在五碳糖再生过程中起作用。在此过程中,PRK消耗光合作用光反应的产物ATP,将其γ-磷酸基团转移到5-磷酸核酮糖(Ru5P)上,催化生成1,5-二磷酸核酮糖(RuBP)及二磷酸腺苷(ADP)。RuBP作为固定二氧化碳的酶Rubisco的底物,起始光合作用的二氧化碳固定过程。关于PRK结构和催化机制的研究早在1998年就有报道,但这些研究都是基于非放氧光合细菌紫细菌来源的PRK(Rhodobacter spheaeroides PRK,RsPRK)进行的。放氧光合生物来源的PRK与RsPRK在蛋白序列上的同源度极低,只有17.1%,并且它们的寡聚状态和调控方式方面也截然不同。RsPRK形成同源八聚体,而来源于放氧光合生物的PRK一般以同源二聚体形式存在。且在每个单体中,N端都含有一对受氧化还原调控的半胱氨酸,处于还原态的PRK为活性状态,氧化态的PRK为失活状态;而RsPRK中没有这对半胱氨酸,因而不受氧化还原调控。最近,有研究揭示了蓝藻PCC 6301的氧化态PRK结构,以及拟南芥和莱茵衣藻的还原态 PRK结构。这些结构说明了放氧光合生物PRK的二体形成形式以及受氧化还原调控的半胱氨酸的位置。在还原态PRK中,两个半胱氨酸分开较远距离,氧化态PRK中两个半胱氨酸形成了 二硫键。在激酶中,通常包含一段相对保守的motif,由序列G-x(4)-GK-[TS](Gly-任意四个氨基酸-Gly-Lys-Thr或Ser)组成,这段序列被称为P-loop,通常参与激酶中的ATP结合。在PRK中,一个半胱氨酸(C17)正位于P-loop结构域,当与另一个半胱氨酸(C56)形成二硫键后,可能破坏了PRK中ATP的结合位点,从而导致氧化态PRK失去活性。
在光合作用暗反应阶段的三碳糖还原过程中,甘油醛-3-磷酸脱氢酶(GAPDH)可以将1,3-二磷酸甘油酸还原为甘油醛-3-磷酸,这个过程要消耗光合作用光反应产物NADPH。根据序列,GAPDH通常可分为两种类型,分别是GapA和GapB。它们在叶绿体中可以组装成A4和A2B2两种四聚体形式。在蓝藻和真核藻类中,GAPDH只以A4-GAPDH形式存在。这种四聚体状态稳定,具有持续活性,其活性的调节主要通过与CP12形成复合物,此时活性受到抑制。A2B2异源四聚体形式的GAPDH则主要受到TRX的氧化还原调控,但是在部分陆生植物中,其活性也能通过与CP12形成复合物而被抑制。目前已发现CP12存在于多种放氧光合生物中。经典的CP12在序列的中间部分含有一段高度保守片段“AWD_VEE”。还原态CP12是无序蛋白,没有固定结构;氧化态CP12则形成稳定的三维构象,并能够进一步与GAPDH、PRK形成复合物。当植物接受光照时,TRX会还原CP12,还原态的CP12失去二级结构,无法维持复合物的组装,导致复合物解聚。之前的研究表明GAPDH和PRK形成复合物后,PRK的活性受到抑制,但是从复合物中解聚出的PRK能够被快速激活,而游离的氧化态PRK恢复活性相对较慢。截至我们的工作完成之前,这一现象的机制尚不明确。之前的研究指出GAPDH/CP12/PRK复合物的意义不仅在于抑制GAPDH和PRK的活性,其更重要的作用在于植物处于黑暗条件时,为PRK和GAPDH提供储蓄库,保护两种酶在不发挥功能时维持其结构稳定,不被细胞降解,当植物再次受到光照时,复合物解聚使得GAPDH与PRK能快速恢复活性,起始卡尔文循环,保证光合作用高效进行。
最近,来源于蓝藻的GAPDH/CP12/PRK复合物整体分辨率的电镜结构已被报道。但该结构中由于PRK与CP12界面处分辨率比较低,不能确定两种蛋白的具体结合细节。另外,尽管目前已有文章报道了放氧光合生物的PRK结构,但是一直没有PRK结合底物及催化辅因子的结构,因而对于ATP和Ru5P与PRK的结合方式,以及PRK的催化机制仍不清楚。
我们解析了蓝藻来源的PRK(Synechococcus elongatusPRK,SePRK)的晶体结构,结构中SePRK结合了ADP及PRK底物5-磷酸核酮糖结构相近的小分子葡萄糖-6-磷酸(G6P),以及拟南芥来源的GAPDH/CP12/PRK复合物(AtGAPDH/CP12/PRK)晶体结构。这两个结构展示了PRK活性位点细节,以及参与PRK-CP12相互作用的结构域。此外,我们还解析了拟南芥来源的还原态和氧化态PRK(Arabidopsis thaliana reduced PRK,AtPRKred和Arabidopsisthaliana oxidized PRK,AtPRKox)结构。通过结构分析,我们构建了大量的PRK突变体,测定了这些突变体相比于野生型PRK的活性变化,以及与底物/辅因子结合能力的差别。结合这些生化实验结果,我们揭示了PRK的催化及调控机制:在氧化态PRK中,ATP结合位点遭到破坏导致PRK失活;而复合物的形成使得CP12结合在了Ru5P位点上,从而导致PRK失活。我们的结构和功能研究大幅提高了人们对PRK催化机制和卡尔文循环的氧化还原调控机制的理解,同时,我们构建的突变体也表现出与野生型PRK不一样的催化活性,可以进一步应用于采用生物技术手段改造作物,达到提高光合作用暗反应阶段的固碳效率,提高作物产量的目的。
发明内容
本发明首先涉及一组拟南芥植物的磷酸核酮糖激酶(AtPRK)的几种突变体,所述的突变体为:在如SEQIDNO.1所示的野生型AtPRK蛋白的序列基础上,突变了如下位点形成的突变体:
(1)D14A、S15A、K19A、S20A、W156A中的任一或任意组合;
(2)D58A、H61A、R65A、R68A、K69A、Y104F、H106A中的任一或任意组合;
或(3)上述两组突变位点的任意组合。
其中第(1)组突变涉及ATP亲和力的变化,第(2)组突变涉及Ru5P亲和力的变化。
优选的,所述的突变体为突变体1,其序列为:在如SEQIDNO.1所示的野生型PRK蛋白的序列基础上,突变了如下氨基酸位点:D58A或R65A或Y104F或H106A;
优选的,所述的突变体为突变体2,其序列为:在如SEQIDNO.1所示的野生型PRK蛋白的序列基础上,突变了如下氨基酸位点:K19A或W156A;
优选的,所述的突变体为突变体3,其序列为:在如SEQIDNO.1所示的野生型PRK蛋白的序列基础上,突变了如下氨基酸位点:S15A或S20A;
最优选的,所述的突变体为突变体4,其序列为:在如SEQIDNO.1所示的野生型PRK蛋白的序列基础上,突变了如下氨基酸位点:K69A;
SEQIDNO.1:
TCAQETIVIGLAADSGCGKSTFMRRLTSVFGGAAKPPKGGNPDSNTLISDTTTVICLDDYHSLDRYGRKEQKVTALDPRANDFDLMYEQVKALKNGIAVEKPIYNHVTGLLDPPELIQPPKILVIEGLHPMFDERVRDLLDFSIYLDISNEVKFAWKIQRDMAERGHSLESIKASIEARKPDFDAFIDPQKQYADAVIEVLPTTLIPDDNEGKVLRVRLIMKEGVKYFSPVYLFDEGSTISWIPCGRKLTCSYPGIKFNYEPDSYFDHEVSVLEMDGQFDRLDELIYVESHLSNLSTKFYGEVTQQMLKHADFPGSNNGTGLFQTIVGLKIRDLYEQLIANKATARAEAKA;
编码野生型AtPRK蛋白的核苷酸序列如SEQIDNO.2所示;
SEQIDNO.2:
ATGGGCGCACAAGAAACCATCGTGATCGGACTAGCTGCTGACTCTGGCTGCGGCAAAAGTACCTTTATGCGGAGGCTCACCAGCGTCTTTGGTGGCGCTGCTAAGCCACCAAAAGGCGGGAACCCTGATTCCAACACACTCATCAGCGACACGACCACTGTGATCTGTCTTGATGATTACCATTCTTTGGATAGGTACGGTAGGAAAGAGCAGAAAGTCACCGCTTTGGACCCACGCGCCAATGACTTTGATCTCATGTATGAGCAAGTCAAAGCTCTTAAGAATGGTATAGCCGTCGAGAAACCGATTTATAACCATGTCACTGGACTTCTTGACCCTCCGGAGCTTATTCAGCCTCCTAAGATTCTTGTCATCGAAGGTCTTCACCCAATGTTTGATGAGCGAGTAAGAGACTTACTAGACTTCAGTATCTACTTGGACATTAGCAACGAAGTCAAATTCGCTTGGAAAATTCAGAGGGACATGGCTGAAAGAGGTCACAGTTTGGAGAGCATCAAAGCGAGTATCGAAGCCCGAAAGCCCGACTTCGATGCATTCATCGACCCGCAAAAGCAGTACGCGGATGCGGTCATAGAAGTGCTTCCTACGACTCTGATCCCAGATGACAACGAAGGGAAAGTGTTGAGAGTGAGATTGATAATGAAGGAAGGTGTTAAGTACTTCAGCCCGGTTTACCTGTTCGATGAAGGTTCAACCATCTCGTGGATTCCTTGCGGCCGCAAACTCACTTGCTCGTACCCTGGCATCAAGTTCAACTACGAACCTGACTCCTACTTCGACCATGAGGTATCAGTTTTGGAGATGGATGGACAATTTGATAGACTGGACGAGCTGATTTACGTGGAAAGTCACTTGAGCAACCTCTCGACCAAATTCTACGGAGAAGTCACTCAACAAATGCTCAAACATGCTGATTTCCCGGGTAGCAACAACGGTACTGGTCTTTTCCAAACCATTGTTGGATTGAAGATCAGAGATCTCTATGAGCAGCTCATTGCCAACAAAGCCACTGCTCGTGCAGAAGCTAAAGCC。
本发明还涉及拟南芥磷酸核酮糖激酶同源蛋白的突变体,即来源于不同种属的PRK蛋白,所述突变体的突变位点为拟南芥PRK同源蛋白的如下同源位点所对应的氨基酸:
(1)D14A、S15A、K19A、S20A、W156A中的任一或任意组合;
(2)D58A、H61A、R65A、R68A、K69A、Y104F、H106A中的任一或任意组合;
或(3)上述两组突变位点的任意组合;
所述的同源位点为以AtPRK为基准蛋白确定的氨基酸位点。
优选的,所述的同源蛋白为:
PsPRK:豌豆(Pisum sativum)的磷酸核糖激酶,
SoPRK:菠菜(Spinacia oleracea)的磷酸核糖激酶,
OsPRK水稻(Oryza sativa)的磷酸核糖激酶,
ZmPRK玉米(Zea mays)的磷酸核糖激酶。
所述的同源蛋白的氨基酸序列对应关系如图1所示。
本发明还涉及所述突变体的如下应用:
(1)构建改变卡尔文循环(暗反应阶段)功能的模型植株;
(2)构建植物模型;
(3)构建具有改进的固碳功能的植株;
(4)催化5-磷酸核酮糖(Ru5P)为1,5-二磷酸核酮糖。
附图说明
图1、不同物种的PRK同源蛋白的氨基酸序列对应图。
图2、PRK蛋白发挥催化功能的关键位点结构及相关氨基酸示意图。
图3、PRK蛋白的活性位点。3A、SePRK与ADP及G6P结合的结构;3B、SePRK的表面电势图;3C、模拟结合配体ATP和Ru5P的SePRK表面电势图;3D、SePRK结构中结合的ADP和G6P分子与模拟分析SePRK结合的ATP和Ru5P分子。
图4、PRK蛋白的整体结构,4A、AtPRK的晶体结构;4B、PRK二聚体在晶体结构中的结构叠合图:AtGAPDH/CP12/PRK三元复合物中的PRK、AtPRKred、AtPRKox和SePRK。4C、四种晶体结构中PRK单体的结构比较。
图5、AtGAPDH/CP12/PRK复合物晶体结构
图6、通过等温滴定量热法测量AtPRK野生型和突变体以及野生型SePRK和Ru5P结合亲和力。
图7、通过表面离子共振技术测量野生型和突变型AtPRK以及野生型SePRK的ATP结合亲和力。
具体实施方式
材料:
Gene code:AtPRK:AT1G32060,AtGAPDH:AT3G26650,AtCP12:AT3G62410;SePRK:synpcc7942_0977;
限制性内切酶:Nco1,Xho1(Takara Quickcut),Ssp1(NEB);
T4连接酶(NEB);
T4 DNA聚合酶(Thermo fisher);
Plasmid Mini KitⅠ(200)(OMEGA);
Gel Extraction Kit(200)(OMEGA);
Competent cell:Trans10,BL21(TRANSGEN BIOTECH)
Chelating SepharoseTM Fast Flow(GE Healthcare);
Size exclusion chromatography:Superdex 200(GE Healthcare);
Spectrophotometer U-3900UV/VIS(HITACHI);
Surface plasmon resonance:Biacore 8K(GE Healthcare);SAchip:(Series SGE Healthcare);
Isothermal Titration Calorimetry instrument(Malvern);
Crystallization kit:PEG IONⅡ(Hampton Research);Procomplex(Moleculardimensions)
酶活及配体亲和力测定
PRK酶活测定:酶活测定分析方法利用了PRK催化反应过程中水解产生的ADP与NADH的氧化发生级联反应。PRK利用ATP和Ru5P催化生成ADP和RuBP。丙酮酸激酶催化磷酸烯醇式丙酮酸和ADP生成ATP和丙酮酸,乳酸脱氢酶将丙酮酸和NADH转化为乳酸和NAD+。实验开始之前,先测定NADH浓度与340nm吸光值(A340)之间的关系,将NADH分别定浓度为0mM,0.1mM,0.2mM,0.3mM,0.4mM,利用紫外分光光度计分别测定A340约为0,0.5,1,0.15,0.2,因此确定NADH浓度与A340之间为线性关系,且A340数值为NADH浓度值的5倍。根据A340变化计算出NADH的变化量,NADH的减少量与PRK催化产生的ADP生成量相等,再根据反应时间,最后计算出PRK产物生成速率。
将100mM Tris-HCl,pH 8.0,4mM MgCl2,10mM KCl,10mM DL-二硫苏糖醇(DTT),0.3mM NADH,5U丙酮酸激酶(PK),5U乳酸脱氢酶(LDH),0.5mM磷酸烯醇式丙酮酸(PEP)和1mMATP加入50ul石英比色皿,PRK以终浓度40ng/ml添加到测活体系中并在298K条件下孵育10min,最后加入终浓度为1.2mM的Ru5P起始反应,持续测A340的吸光值10min。反应总体积为50ul,野生型和突变体PRK的测活反应均在298K条件下进行。
ATP亲和力测定:将蛋白野生型AtPRK、突变体D14A、S15A、K19A、S20A、W156A分别与生物素按照1:1的摩尔比孵育30min使之共价结合,再用500ul容量的50kDa浓缩管在4000g离心力作用下离心5min进行浓缩,去除浓缩管下液,加20mM HEPES,pH7.5,100mM NaClbuffer至截留的蛋白浓缩液中稀释到500ul,三次反复浓缩稀释的方法去除多余的生物素。在298K温度下利用表面离子共振技术(SPR)测定ATP与PRK的相互作用。流动buffer为20mMHEPES,pH7.5,100mM NaCl。固定蛋白之前将SA芯片先用含有1M NaCl和5mM NaOH的溶液处理4min,最后将去除游离生物素的蛋白稀释到40ug/ml固定到芯片上。ATP用流动buffer梯度稀释为7.8125uM,15.625uM,31.25uM,62.5uM,125uM,250uM,500uM,1000uM,2000uM,分别流过固定蛋白的芯片,测各流动循环的反应值(response units)。最终通过各ATP浓度流过芯片时的反应值进行数据分析拟合,计算亲和力。
Ru5P亲和力测定:等温滴定量热(ITC)实验在298K条件下进行,使用MicroCaliTC200仪器。将1mM ATP和2mg/ml的PRK蛋白在含有100mM Tris-HCl,pH 8.0,4mM MgCl2,10mM KCl,2mM DTT的缓冲液中混合好放入ITC反应室中孵育10min,再用同样缓冲液稀释的0.5mM Ru5P加入滴定针中,将进样后的滴定针放入反应室中,以750rpm的转速混匀反应室10min,最后以单次滴定体积2ul,样品单次进样时间2s/ul,滴定间隔时间120s的方式逐渐将Ru5P分20次滴入反应室中,测量反应室中的热量变化,最后进行数据拟合分析Ru5P与PRK的亲和力。Ru5P与野生型AtPRK及其突变体D58A,H61A,R65A,R68A,K69A,Y104F,H106A亲和力分别用以上方法测定。。
所有酶活测定和ATP,Ru5P亲和力测定实验都至少重复了两次。
实施例1、突变体的构建及突变体蛋白的表达纯化
1.1、突变体设计
利用聚合酶链式反应(PCR)方法(对应引物如下表1所示)从拟南芥Arabidopsisthaliana和蓝藻Synechococcus elongates PCC7942的基因文库分别扩增得到AtPRK和SePRK基因。扩增产物用琼脂糖电泳分离纯化胶回收之后,与空载体pET-28a一同用限制性内切酶Nco1和Xho1在37℃处理1小时,再用T4连接酶将酶切后的基因片段连接到酶切后的pET-28a载体上,使得基因连接到载体的Nco1和Xho1限制酶切位点之间。每条基因在C末端具有编码6个组氨酸的标签(His-tag)。AtPRK的突变体都是利用重叠PCR(对应引物如下表1所示)方法做的定点突变。将之前扩增得到的野生型PRK基因作为模板,以S15A突变体为例:用AtPRK-pET-28a上游引物5’CCCATGGGCGCACAAGAAACCATCGTGATCGGAC 3’与PRK-S15A下游引物5'TACTTTTGCCGCAGCCCGCGTCAGCAGCTAGTC 3'通过PCR方法得到目的基因上游片段,PCR产物用琼脂糖凝胶电泳分析,确定大小正确后进行切胶回收;再用AtPRK-pET-28a下游引物5'CCGCTCGAGGGCTTTAGCTTCTGCACGAGCAGTG 3'和与PRK-S15A上游引物5’GACTAGCTGCTGACGCGGGCTGCGGCAAAAG 3’通过PCR得到目的基因下游片段。PCR产物用琼脂糖凝胶电泳分析并进行切胶回收;最后以回收的两次PCR得到的上、下游片段为模板,加入AtPRK-pET-28a的上、下游引物,通过PCR得到完整的S15A突变体基因,再用琼脂糖凝胶电泳分析并切胶回收。回收产物用限制性内切酶Nco1和Xho1处理,再将得到的突变体基因用T4链接酶连接到pET-28a载体中。其余突变体构建过程与S15A构建过程一致。
为了获得不含His-tag的蛋白用于组装AtGAPDH/CP12/PRK复合物,通过PCR技术利用下表1所示的引物从拟南芥cDNA文库中扩增AtPRK、AtGAPDH和AtCP12基因。以AtPRK为例,用引物表1中AtPRK-pMCSG7引物进行基因扩增的产物通过琼脂糖电泳和胶回收后,用不依赖于连接反应的高效克隆方法(LIC)连接到线性化后的载体pMCSG7中。将胶回收得到的AtPRK目的基因与空载体pMCSG7分别用Ssp1在37℃酶切10小时,再将酶切产物进行琼脂糖电泳鉴定和切胶回收;在回收的酶切产物目的基因片段AtPRK中加dGTP(脱氧鸟苷三磷酸)和T4 DNA聚合酶,在pMCSG7载体酶切产物中加dCTP(胞嘧啶三磷酸)和T4 DNA聚合酶,在25℃条件下分别处理30min,再用75℃水浴20min使T4 DNA聚合酶失活;最后用T4连接酶将AtPRK目的基因连入pMCSG7载体中。pMCSG7载体上游含有一个带有烟草蚀纹病毒(TEV)酶切位点的His-tag,使得蛋白表达之后N端带有一个His-tag,且该tag可以用TEV酶切掉。
表1、本发明所涉及的构建各个突变的重叠PCR引物表
重组质粒利用大肠杆菌Escherichia coli Top10进行扩增。所有重组后的基因全部进行了DNA测序进行检测。测序正确的重组质粒转化到Escherichia coli BL21(DE3)中进行表达。
1.2、突变体蛋白纯化
由于pET-28a载体含有卡那抗生素抗性,野生型及突变体AtPRK-pET-28a转化的E.coli BL21细胞用含有50ug/ml卡那霉素的Lysogeny broth培养基在37℃条件下培养4h;pMCSG7转化的E.coli BL21细胞用含有100ug/ml氨苄青霉素的LB培养基在37℃条件下培养4h。后续表达和纯化步骤全部相似,描述如下。
当E.coli BL21细胞密度在波长600nm光下吸光值(OD600)达到0.8时,向培养基添加终浓度1mM异丙巯基-β-半乳糖苷(IPTG)诱导,继续在30℃培养16h。然后用8,000×g离心力离心培养基10min,收集沉淀中的细胞。将沉淀用buffer A(20mM Tris-HCl,pH 8.0,500mM NaCl,20mM imidazole)重悬并超声破碎。再将细胞裂解液在4℃下用18,000×g离心力离心40min。将含有目的蛋白的离心上清液流入已用buffer A平衡过的镍亲和层析柱,上样结束后用buffer B(20mM Tris-HCl,pH 8.0,500mM NaCl,50mM imidazole)洗掉杂蛋白。最后用buffer C(20mM Tris-HCl,pH 8.0,500mM NaCl,250mM imidazole)洗脱目的蛋白。带有C端His-tag的重组蛋白再直接用分子排阻层析法(Superdex 200,10/300GL柱,GEhealthcare)进行进一步分离纯化,用buffer D(20mM Hepes,pH 7.5,100mM NaCl)进行平衡和洗脱。
1.3、AtGAPDH/CP12/PRK复合物纯化
重组的AtPRK、AtGAPDH和AtCP12蛋白由于构建到了pMCSG7载体中,含有氨苄青霉素抗性,因此转化后的E.coli BL21细胞使用含有100ug/ml氨苄青霉素抗生素的LB培养基培养,在37℃条件下培养4h。当E.coli BL21细胞密度在波长600nm光下吸光值(OD600)达到0.8时,向培养基添加终浓度1mM异丙巯基-β-半乳糖苷(IPTG)诱导,继续在30℃培养16h。然后用8,000×g离心力离心培养基10min,收集沉淀中的细胞。将沉淀用buffer A(20mMTris-HCl,pH 8.0,500mM NaCl,20mM imidazole)重悬并超声破碎。再将细胞裂解液在4℃下用18,000×g离心力离心40min。将含有目的蛋白的离心上清液流入已用buffer A平衡过的镍亲和层析柱,上样结束后用buffer B(20mM Tris-HCl,pH 8.0,500mM NaCl,50mMimidazole)洗掉杂蛋白。最后用buffer C(20mM Tris-HCl,pH 8.0,500mM NaCl,250mMimidazole)洗脱目的蛋白。由于构建在pMCSG7载体的AtPRK、AtGAPDH和AtCP12蛋白在N端都具有His-tag,将三种蛋白分别与TEV酶在4℃孵育过夜,消化好的目的蛋白再次流过镍亲和层析柱,再用分子排阻层析分离纯化,得到没有tag的AtPRK、AtGAPDH和AtCP12蛋白。为了获得AtGAPDH/CP12/PRK复合物,AtGAPDH先与2mM NAD在4℃孵育5h,再将AtGAPDH、AtPRK和AtCP12按照亚基摩尔比8:4:4混合,由于PRK和CP12需要处于氧化态才能形成复合物,CuSO4作为氧化剂以0.01mM浓度加入蛋白混合物中,在4℃孵育12h。最终,体外组装的复合物用Superdex 200 10/300GL柱(GE Healthcare)通过分子排阻层析分离纯化,平衡buffer为20mM HEPES,pH7.5,100mM NaCl。
每种纯化得到的蛋白都通过SDS凝胶电泳分析,并用基质辅助激光电离/飞行时间质谱(MALDI-TOF-MS)做肽指纹图谱分析进行鉴定。
实施例2、PRK的活性位点及突变体功能分析
2.1、PRK的活性位点及ATP,Ru5P的结合
之前所有报道的PRK结构都仅有蛋白分子,没有配体结合,缺少PRK与ATP或Ru5P结合的细节。为了指认底物和辅因子的结合方式,我们尝试将AtPRK和SePRK与ATP和Ru5P以及与其结构相似的小分子化合物共晶,最终得到了结合有ADP和G6P的SePRK结构。尽管我们用ATP与SePRK共晶,但在这个结构中只有ADP清晰可见,ATPγ位置上的磷酸基团(γ-P)可能在结晶过程中被水解掉了。
SePRK结构表面有两个窄凹槽相互垂直呈“L”形,这两个凹槽主要由带正电荷氨基酸组成(图3B)。每个凹槽各被一个配体占据;ADP结合的凹槽(命名为ATP结合位点)主要由P-loop(15DSGCGKST22)及其附近结构域构成。ADP的α-磷与T22相互作用,β-磷与S21(对应AtPRK S20)之间形成氢键相互作用。此外,腺苷环与W140(对应AtPRK W156)平行,且与T304形成氢键(图3A)。在第二个结合口袋中,G6P分子的磷酸基团与R52(对应AtPRK R68),R163(对应AtPRK R179)形成氢键,R49(对应AtPRK R65),Y88(对应AtPRK Y104)和H90(对应AtPRK H106)固定住了G6P的羟基(图3A)。
在结构基础上,我们进一步用Autodock Vina程序将Ru5P和ATP分别与SePRK进行了分子对接计算(图3C)。在预测的SePRK结合Ru5P,ATP的模型中,Ru5P的磷酸根与G6P磷酸根结合位置相似,Ru5P位点主要由D42(AtPRK D58),R49(AtPRK R65),R52(AtPRK R68),Y88(AtPRK Y104),H90(AtPRK H106)和R163(AtPRK R179)等氨基酸残基构成(图2)。ATP结合预测结果说明P-loop上的S16(AtPRK S15),K20(AtPRK K19),S21(AtPRK S20)固定了γ-P(图2)。以上这些残基均在放氧光合生物PRK中高度保守(图1),说明这些氨基酸对PRK发挥功能具有重要作用。
2.2、AtPRK中参与结合配体和催化的重要氨基酸
叠合对比分析显示AtPRKred与SePRK的整体结构几乎一致(图4),这意味着两个酶结合ATP和Ru5P的位点也相似。基于这些发现,我们对AtPRK中可能作为底物结合位点和具有催化活性的氨基酸进行突变,并测定酶活。酶活实验分析表明D58A,Y104F和H106A三个突 变体几乎完全失去活性,S15A,K19A,S20A,R65A和W156A相比于野生型,催化活性降低明显, 而K69A突变体相较于野生型活性增强。
AtPRK中的S15,K19,S20和W156分别对应SePRK中的S16,K20,S21和W140,在SePRK中这四个氨基酸参与ATP的结合。通过表面等离子共振技术(SPR)测定AtPRK与ATP的亲和力发现K19和W156分别突变成Ala之后,ATP亲和力明显下降,由于K19侧链带正电荷利于稳定ATP带负电荷的磷酸基团,因而K19对ATP结合非常重要,这一结果与SePRK结构观察相同。S15A和S20A突变体对ATP结合影响较小,这两个丝氨酸与ATPγ-P相互作用,它们主要功能是固定ATPγ-P取向和位置,从而保证了磷酸根基团的转移,完成催化反应。
此外,结构分析还显示AtPRK中D58,R65,Y104和H106(SePRK中的D42,R49,Y88和H90)参与Ru5P结合。我们用等温滴定量热法(ITC)测定了野生型和突变体D58A,R65A,Y104A和H106A对Ru5P的亲和力,发现D58,R65,Y104,H106突变后Ru5P亲和力下降,说明这四个氨 基酸与Ru5P结合相关(图6)。
由于我们在测活过程中使用的Ru5P浓度1.2mM,远高于AtPRK结合Ru5P的KD值,即所有突变体结合底物都能达到饱和,说明测得的突变体酶活差异与蛋白自身催化效率有关。因此,D58A,H106A突变体几乎完全失活不是因为Ru5P亲和力下降,而是因为催化能力受到影响,说明D58和H106是直接参与催化的氨基酸。基于我们的SePRK分子对接计算结果,AtPRK中D58和H106位于ATP和Ru5P交汇的位置,与ATP的γ-P和Ru5P C1位的羟基距离很近(图4A),因而D58和H106可能通过直接或间接参与亲核攻击来起始催化反应。
另外,我们还发现位于Ru5P磷酸基团结合位点附近的K69,将其突变为Ala之后,并不影响Ru5P的亲和力,但酶活有小幅度提高,说明K69突变为Ala后PRK的催化能力有所提高。若将PRK的K69A突变体转入植物体内,其转化Ru5P为RuBP的速率可能高于野生型PRK,可以更加高效的为CO2固定提供底物,为糖类合成提供更多前体,从而加速植物生长。
本发明针对AtPRK的活性位点进行了一系列突变,所述的各个突变体的功能活性测定结果如下表2所示表2、各个突变体的功能活性测定
实施例3、AtGAPDH/CP12/PRK复合物的结构分析
3.1、AtGAPDH/CP12/PRK复合物的结构
AtGAPDH/CP12/PRK结构呈类似于纺锤状,由两个A4-GAPDH四聚体,两个氧化态PRK二体,四个CP12单体组成(图5)。两个A4-GAPDH四聚体分别位于纺锤的两端顶点,PRK组成了纺锤形状的侧边,CP12的作用是将PRK和GAPDH连接在一起。在复合物中,氧化态的CP12形似一个钩子,由三段α螺旋构成。N端螺旋(G6-T21)和中心螺旋(E30-G56)形成了发夹结构,组成CP12 N端结构域,两段螺旋之间由N端两个半胱氨酸形成的二硫键连接(C22-C31),CP12的保守序列(34AWD_VEE40)位于中心螺旋的N端部分。CP12的C端相较于N端的长直结构更接近球状,由一段较短的C端螺旋和loop构成,二者之间由C端两个半胱氨酸形成的二硫键连接(C64-C73)。CP12的N端和C端结构域分别与PRK及GAPDH相互作用。
3.2、PRK中参与复合物形成的重要氨基酸
复合物中每个GAPDH四聚体通过两个CP12单体连接两个PRK二体。CP12的C端loop插入两个GAPDH单体的界面处,与之前报道的GAPDH-CP12 CTD结构一致。CP12的N端与PRK相互作用,中心螺旋位于PRK表面上的长凹槽中,该凹槽由P-loop(D14-T21),D64-Q71和R160-L169三个结构域组成,它们分别构成了凹槽的底部和侧边。值得注意的是,这些结构域亦参与PRK活性位点构成。这三个结构域形成的带有正电荷的凹槽围绕着CP12的保守结构域34AWD_VEE40。这一结构域中的酸性氨基酸E40和D36分别与PRK表面的碱性氨基酸R65和R68结合。将R65或R68突变为Ala之后,PRK失去与GAPDH,CP12形成复合物的能力,说明这两个氨基酸在稳定PRK-CP12结合中起重要作用。
通过结构分析,我们还发现AtPRKred中包含二硫键的P-loop构象不影响CP12的结合,意味着还原态PRK可能也能如氧化态PRK一样参与复合物形成,这就能解释为什么从复合物中解聚出的PRK能够比游离的氧化态PRK更快速地被激活。为了验证这一假设,我们构建了C17S PRK突变体,因为C17S无法与C56形成二硫键,因而该突变体无法被氧化一直维持还原态。将该突变体与GAPDH,氧化态CP12孵育,分子筛结果显示PRK C17S突变体也能与氧化态PRK一样参与形成GAPDH/CP12/PRK复合物。
此外,酶活测定结果显示,C17S突变体与野生型PRK催化反应速率一致。C17S突变体无法受到氧化调控,因此可以持续维持PRK处于活性状态。而在黑暗条件下叶绿体处于氧化状态时,由于C17S也能与GAPDH,CP12形成复合物,使得该突变体在氧化环境中通过形成复合物GAPDH/CP12/PRK得以储存和保护。当植物再次受到光照时GAPDH/CP12/PRK解聚,由于C17S PRK不需再经过还原反应激活,从复合物释放出的突变体PRK可立即恢复活性。因此当植物受到光照时,C17S突变体起始反应的速度会快于野生型PRK,从而使得卡尔文循环能更快启动,提高光合作用效率。
3.3、PRK的催化和调控机制
将还原态AtPRKred和氧化态AtPRKox结构进行叠合对比发现,它们仅在P-loop区域有构象上的差异。进一步比对SePRK和AtPRKox结构能清楚发现ATP位点在AtPRKox中被破坏掉了。然而,Ru5P位点距离P-loop较远,不受P-loop构象变化影响。这些结构说明氧化态PRK失活是因为它失去了结合ATP的能力,激活(还原态)和失活(氧化态)状态的开关就是P-loop的构象变化,这一状态转换并不影响Ru5P与PRK的结合。
除了氧化还原调控,PRK另一个重要的调控机制就是可逆地形成GAPDH/CP12/PRK复合物。在复合物中,CP12结合在它们的活性位点,使得GAPDH和PRK均处于失活状态,其中CP12中心的α螺旋恰好结合在Ru5P位点。CP12的两个酸性氨基酸D36和E40插入了Ru5P位点,D36的位置刚好阻碍了Ru5P结合,而这两个酸性氨基酸在复合物中分别与PRK的R65和R68形成氢键。因此,我们的结果说明CP12刚好结合在PRK中的Ru5P位点,阻止了Ru5P的结合,从而揭示了复合物中PRK活性降低的原因。
值得注意的是,我们发现ATP结合PRK是Ru5P结合的先决条件。ITC实验中,如果用Ru5P滴定AtPRK自身无法检测到结合信号,而用Ru5P滴定AtPRK与ATP孵育的混合物,则能检测到可靠的Ru5P结合结果(图6)。ATP与PRK孵育后会增强Ru5P亲和力,但Ru5P与PRK孵育不会影响ATP的结合(图7)。这个实验结果说明PRK先要结合ATP,引起构象变化之后再与Ru5P结合。我们的结果与之前的报道认为PRK结合底物遵循顺序机制是一致的,并进一步指出在顺序机制中,PRK先结合ATP,再结合Ru5P。
最后需要说明的是,以上实施例仅用作帮助本领域技术人员理解本发明的实质,不用做限定本发明的权利要求。
SEQUENCE LISTING
<110> 中国科学院生物物理研究所
<120> 一种拟南芥磷酸核酮糖激酶的突变体
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 351
<212> PRT
<213> Arabidopsis thaliana
<400> 1
Thr Cys Ala Gln Glu Thr Ile Val Ile Gly Leu Ala Ala Asp Ser Gly
1 5 10 15
Cys Gly Lys Ser Thr Phe Met Arg Arg Leu Thr Ser Val Phe Gly Gly
20 25 30
Ala Ala Lys Pro Pro Lys Gly Gly Asn Pro Asp Ser Asn Thr Leu Ile
35 40 45
Ser Asp Thr Thr Thr Val Ile Cys Leu Asp Asp Tyr His Ser Leu Asp
50 55 60
Arg Tyr Gly Arg Lys Glu Gln Lys Val Thr Ala Leu Asp Pro Arg Ala
65 70 75 80
Asn Asp Phe Asp Leu Met Tyr Glu Gln Val Lys Ala Leu Lys Asn Gly
85 90 95
Ile Ala Val Glu Lys Pro Ile Tyr Asn His Val Thr Gly Leu Leu Asp
100 105 110
Pro Pro Glu Leu Ile Gln Pro Pro Lys Ile Leu Val Ile Glu Gly Leu
115 120 125
His Pro Met Phe Asp Glu Arg Val Arg Asp Leu Leu Asp Phe Ser Ile
130 135 140
Tyr Leu Asp Ile Ser Asn Glu Val Lys Phe Ala Trp Lys Ile Gln Arg
145 150 155 160
Asp Met Ala Glu Arg Gly His Ser Leu Glu Ser Ile Lys Ala Ser Ile
165 170 175
Glu Ala Arg Lys Pro Asp Phe Asp Ala Phe Ile Asp Pro Gln Lys Gln
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Tyr Ala Asp Ala Val Ile Glu Val Leu Pro Thr Thr Leu Ile Pro Asp
195 200 205
Asp Asn Glu Gly Lys Val Leu Arg Val Arg Leu Ile Met Lys Glu Gly
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Val Lys Tyr Phe Ser Pro Val Tyr Leu Phe Asp Glu Gly Ser Thr Ile
225 230 235 240
Ser Trp Ile Pro Cys Gly Arg Lys Leu Thr Cys Ser Tyr Pro Gly Ile
245 250 255
Lys Phe Asn Tyr Glu Pro Asp Ser Tyr Phe Asp His Glu Val Ser Val
260 265 270
Leu Glu Met Asp Gly Gln Phe Asp Arg Leu Asp Glu Leu Ile Tyr Val
275 280 285
Glu Ser His Leu Ser Asn Leu Ser Thr Lys Phe Tyr Gly Glu Val Thr
290 295 300
Gln Gln Met Leu Lys His Ala Asp Phe Pro Gly Ser Asn Asn Gly Thr
305 310 315 320
Gly Leu Phe Gln Thr Ile Val Gly Leu Lys Ile Arg Asp Leu Tyr Glu
325 330 335
Gln Leu Ile Ala Asn Lys Ala Thr Ala Arg Ala Glu Ala Lys Ala
340 345 350
<210> 2
<211> 1053
<212> DNA
<213> Arabidopsis thaliana
<400> 2
atgggcgcac aagaaaccat cgtgatcgga ctagctgctg actctggctg cggcaaaagt 60
acctttatgc ggaggctcac cagcgtcttt ggtggcgctg ctaagccacc aaaaggcggg 120
aaccctgatt ccaacacact catcagcgac acgaccactg tgatctgtct tgatgattac 180
cattctttgg ataggtacgg taggaaagag cagaaagtca ccgctttgga cccacgcgcc 240
aatgactttg atctcatgta tgagcaagtc aaagctctta agaatggtat agccgtcgag 300
aaaccgattt ataaccatgt cactggactt cttgaccctc cggagcttat tcagcctcct 360
aagattcttg tcatcgaagg tcttcaccca atgtttgatg agcgagtaag agacttacta 420
gacttcagta tctacttgga cattagcaac gaagtcaaat tcgcttggaa aattcagagg 480
gacatggctg aaagaggtca cagtttggag agcatcaaag cgagtatcga agcccgaaag 540
cccgacttcg atgcattcat cgacccgcaa aagcagtacg cggatgcggt catagaagtg 600
cttcctacga ctctgatccc agatgacaac gaagggaaag tgttgagagt gagattgata 660
atgaaggaag gtgttaagta cttcagcccg gtttacctgt tcgatgaagg ttcaaccatc 720
tcgtggattc cttgcggccg caaactcact tgctcgtacc ctggcatcaa gttcaactac 780
gaacctgact cctacttcga ccatgaggta tcagttttgg agatggatgg acaatttgat 840
agactggacg agctgattta cgtggaaagt cacttgagca acctctcgac caaattctac 900
ggagaagtca ctcaacaaat gctcaaacat gctgatttcc cgggtagcaa caacggtact 960
ggtcttttcc aaaccattgt tggattgaag atcagagatc tctatgagca gctcattgcc 1020
aacaaagcca ctgctcgtgc agaagctaaa gcc 1053
Claims (3)
1.一种拟南芥植物的磷酸核酮糖激酶(AtPRK)的突变体,所述的突变体为:在如SEQ IDNO.1所示的野生型AtPRK蛋白的序列基础上,突变了如下位点形成的突变体:
(1)S15A、K19A、S20A、W156A中的任一或任意组合;
(2)D58A、H61A、R65A、R68A、K69A、Y104F、H106A中的任一或任意组合;
或(3)上述两组突变位点的任意组合。
2.根据权利要求1所述的突变体,其特征在于,所述的突变体序列为:
在如SEQ ID NO.1所示的野生型PRK蛋白的序列基础上,突变了如下氨基酸位点:D58A或R65A或Y104F或H106A;
或在如SEQ ID NO.1所示的野生型PRK蛋白的序列基础上,突变了如下氨基酸位点:K19A或W156A;
或在如SEQ ID NO.1所示的野生型PRK蛋白的序列基础上,突变了如下氨基酸位点:S15A或S20A;
或在如SEQ ID NO.1所示的野生型PRK蛋白的序列基础上,突变了如下氨基酸位点:K69A。
3.权利要求1或2任一所述突变体的如下应用:
(1)构建改变卡尔文循环功能的模型植株;
(2)构建植物模型;
(3)构建具有改进的固碳功能的植株;
(4)催化5-磷酸核酮糖(Ru5P)为1,5-二磷酸核酮糖。
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