【作者】 李红；

【导师】 刘永军；

【作者基本信息】 山东大学 ， 物理化学， 2020， 博士

【摘要】 酶参与催化生命体内的各种代谢反应。相比于传统催化剂,酶的显著优点包括高效性、催化反应专一性以及反应条件温和等。随着酶学的不断发展,酶已经被广泛应用于医药、食品、轻工、环保以及相关科研领域。近年来,随着化学手段、结构生物学、生物信息学和基因操作等技术的发展,科研工作者可以对酶分子进行修饰、改造、设计和开发,使其满足医药和工农业的需求。因此,深入研究酶的结构、性质、功能和催化机理不仅有助于阐明生命现象的本质、理解酶的生物学功能,对于拓展酶的应用和发展酶工程也具有十分重要的意义。在酶学研究中,使用实验方法可以得到酶促反应的反应速率、酶蛋白的晶体结构数据、动力学参数、突变结果等数据。而理论计算则能够给出实验上难以捕获的过渡态和中间体结构,以及反应过程中所涉及的能量学信息等,对实验结果进行补充和解释。随着计算机技术和算法开发的不断发展,计算化学在大尺度生物化学体系的研究中已经成为一个重要的工具。本论文使用分子对接方法、分子动力学模拟以及量子力学与分子力学结合(QM/MM)方法对几类非血红素氧化酶体系进行了系统的理论研究。本文所涉及到的氧化酶均含有金属辅酶因子。由于金属酶分布广泛,并且金属离子的电子结构较为复杂,关于金属酶的理论研究是酶催化领域的研究热点也是研究难点。本论文旨在探究酶促反应过程涉及到的关键问题,例如,确定底物在酶活性位点的可能结合模式、得到过渡态和中间体结构、明确最优反应路径以及探究关键残基和配体在催化反应中的作用等。计算结果可以从微观上阐明酶催化反应机制,为理解酶的生物学功能和酶的应用奠定理论基础。本论文的主要研究内容包括:(1)异腈基合成酶ScoE催化反应机理的理论研究最近的结构分析和生化实验表明,来自蓝藻链霉菌的ScoE酶属于非血红素铁/α-酮戊二酸(Fe/α-KG)依赖的脱羧酶,可以通过去饱和以及脱羧反应催化异腈基团的形成。这一发现为异腈基的形成提供了一种新的机制。IsnA、XnPvcA或者AmbI1/AmbI2这类异腈基合成酶,通过引入额外的碳单元将R-CH(-NH2)-CO2-转化为R-CH(-N≡C)-CO2-。ScoE酶则是通过氧化脱羧催化R-NH-CH2-CO2-转化为R-N≡C。为了探索ScoE酶的催化反应机理,基于高分辨率晶体结构,我们构建了酶-底物复合物模型并进行了一系列的QM/MM计算。计算结果表明,ScoE酶催化反应包含了两个非耦合部分:去饱和部分和脱羧部分。高价铁氧基团引发的去饱和反应包括两个连续的氢抽取步骤,这与其它非血红素Fe/α-KG依赖酶催化的去饱和过程类似。在第二个阶段,底物上的H首先被铁氧基团抽取形成底物自由基,接下来的自由基脱羧非常容易。但是,之前提出的羟基化中间体的脱羧过程却很难发生。结果表明,电子从底物转移到铁中心是降低脱羧能垒的关键因素。因此,中心铁离子不仅负责氢抽取,还在脱羧过程中充当电子中继站。此外,研究发现脱羧过程是一个质子耦合电子转移过程,其中R310等残基组成的氢键网络发挥了关键作用。在整个反应过程中,C-N去饱和过程涉及到的氢抽取是反应决速步,两条路径互为竞争关系,对应的能垒分别为17.6和16.9 kcal/mol,这与实验估计值(17.9～18.1 kcal/mol)—致。这些结果为理解异腈基的生物合成以及非血红素Fe/α-KG依赖酶催化的氧化脱羧反应提供了理论基础。(2)诺加霉素合成酶SnoK和SnoN催化反应机理的研究来自链霉菌属的非血红素Fe/α-KG依赖酶SnoK和SnoN酶参与了诺加霉素(一种蒽环类抗生素)的生物合成。虽然二者具有相似的活性位点,但是却具有不同的催化功能,SnoK负责催化碳环化反应而SnoN酶负责催化羟基异构化。为了阐明两种酶的催化反应机制,我们使用分子对接方法、分子动力学模拟以及QIV/MM方法进行了一系列的计算研究。结果表明,二者催化的反应均发生在五重态势能面上。对于SnoK酶而言,整个反应过程包括两个氢抽提步骤和一个自由基加成步骤,其中自由基加成为决速步,对应的反应能垒为21.7 kcal/mol。计算结果表明,D106氨基酸残基参与了活性位点中氢键网络的构建,该氢键网络对于底物的定位发挥了至关重要的作用。此外,结果显示SnoN酶仅负责中间体的氢抽取过程,目前的计算并没有发现合适的残基可以为底物自由基供氢。这一结果证实了实验猜测,即可能是一种细胞还原剂或者另外一种酶蛋白为底物提供氢原子。另外,我们的对接结果与前人的结构分析吻合,即两种酶的催化特异性是由于底物在铁氧基团附近定位的微小差异引起的。以上计算结果揭示了 SnoK酶和SnoN酶的反应机理,有助于蒽环类抗生素生物合成酶的工程化研究。(3)链脲霉素合成酶SznF催化反应机理的研究链脲霉素是一种来自细菌的含有N-亚硝基脲基团的天然产物,既能够诱发疾病又可以作为重要的癌症化疗药物。最近的实验研究展示了链脲霉素的完整生物合成路径。其中,SznF酶负责催化Nω-甲基-L-精氨酸发生氧化重排,形成N-亚硝基脲中间体。尽管实验上已经对链脲霉素生物合成路径中涉及到的酶结构及其生物学功能进行了大量的研究工作,但是对于SznF酶的催化反应机理仍然不确定。以高分辨率晶体结构为基础,我们构建了酶-O2-底物复合物模型,并进行了动力学模拟和一系列QM/MM计算研究。结果表明,底物与中心铁离子存在两种结合模式,均以双配位的形式与铁离子结合,中心铁离子呈现六配位稳定模式。自旋密度数值表明,反应物中底物和氧气同时被活化。酶促反应共包含五个步骤:近端氧抽取底物上的氢原子,远端氧进攻底物形成桥联中间体、Op-Od键和Nε-Cε键断裂生成NO中间体、随后Fe-OH抽取底物上的氢原子以及NO进攻底物形成N-亚硝基脲产物。计算结果显示,SznF酶催化N-亚硝基脲形成过程中涉及到一个具有自由基性质的NO中间体。Fe-OH复合物单元抽氢步骤为决速步,所对应的能垒为21.0 kcal/mol。通过计算研究,揭示了 SznF酶催化生成N-亚硝基脲基团的反应机理,为深入理解链脲霉素生物合成路径提供了理论基础。(4)槲皮素2,4-双加氧酶QueDFLA催化反应机理研究来自链霉菌属的槲皮素2,4-双加氧酶属于单cupin家族。QueDFLA酶使用镍离子作为活性位点辅因子催化槲皮素的氧化裂解。尽管铁离子可以作为大多数双加氧酶的辅酶因子,但是对于QueDFLA酶来说,铁离子却显示出较低的反应活性。为了理解Ni-QueDFLA酶的反应机理和双氧的活化机制,我们使用QM/MM方法阐明反应细节以及特殊的活化机制。计算结果表明,双氧与中心镍离子存在两种结合模式,并且这两种结合模式可以互相转化。由于镍离子的d轨道与双氧以及槲皮素底物的p轨道之间存在重叠,电子可以从底物经过中心镍离子转移到双氧。因此,双氧和槲皮素通过与镍离子的结合而被同时活化。计算结果表明,整个反应发生在三重态势能面上,一共包括四个基本步骤。其中,Op-Od键沿着镍中心旋转这一非化学步骤被认为是决速步,对应的自由能垒为19.9 kcal/mol。NBO分析结果表明,正是由于Op与镍离子的配位变化导致了该过程对应较高的能垒。另外,由于镍离子同时活化底物和双氧,因此五元环中间体的形成和裂解是非常容易的。在这一过程中,O-O键的裂解与两个C-C键的裂解协同发生。当铁离子作为辅因子时,反应过程中的决速步变成五元环中间体裂解这一步,对应的自由能垒为30.3 kcal/mol。这项研究揭示了 QueD的详细反应机理,有助于理解其它含镍酶的催化机理。本论文的特色和创新之处:(1)确定了异腈基合成酶ScoE的氧化脱羧机制,修正了之前提出的羟基化中间体反应机理,描述了反应过程中涉及到的质子耦合电子转移过程,揭示了中心铁离子在酶促反应中的双重作用,明确了关键残基R310在反应过程中的作用。(2)明确了诺加霉素合成酶SnoK催化的碳环化反应机理和SnoN催化的羟基异构化机理,揭示了关键残基在酶促反应中的重要作用,通过对比两种非血红素Fe/α-KG依赖酶活性位点的结构揭示了二者催化反应特异性的原因。(3)对比分析了两种反应物模型,阐明了 SznF酶的氧气与底物活化机制,计算研究了不同的反应路径,明确了详细催化反应机理,确定了反应过程中出现的NO中间体,分析了关键残基在催化反应中的作用。(4)明确了槲皮素2,4-双加氧酶中双氧与中心镍离子可能的结合模式,阐明了双氧的活化机制,详细描述了完整的催化反应机理,对比了不同金属辅酶因子对QueD酶催化活性的影响。更多还原

【Abstract】 In the body,various kinds of reactions involved in the metabolism are catalyzed by enzymes.Compared with the traditional catalysts,enzymes have many significant advantages,such as high efficiency,high specificity of catalytic reactions and mild reaction conditions.With the continuous development of enzymology,enzymes have been widely used in medicine,food,light industry,environment protection and some related scientific research fields.Furthermore,chemical means,structural biology,bioinformatics and genetic manipulation technologies have developed rapidly in recent years.Researchers can make enzyme more suitable for the pharmaceutical,industrial and agricultural demand by modifying,transforming,designing and developing the enzyme molecules.Therefore,it is necessary to investigate the structure,properties,function and reaction mechanism of enzymes,which will be helpful to clarify the nature of the life,understand the biological function of the enzymes and expand the application field of the enzymes.In the enzymatic reaction researches,experimental methods can be used to obtain some important data,such as the rate values of catalytic reaction,the crystal structures,the kinetic parameters,the mutation results and so on.Theoretical calculation results can provide the structures of the transition states and intermediates,as well as the energetic information involved in the enzymatic reaction,which are difficult to be captured experimentally.Thus the theoretical calculations can be used to supplement and explain the experimental results.With the continuous progress of the computer technology and the algorithm development,the computational chemistry has become an important tool in the investigation of large-scale biochemical systems.In this work,the molecule docking,molecular dynamics simulation and the combination of quantum mechanics/molecular mechanics method(QM/MM)were performed to explore several kinds of non-heme oxidases.All of the oxidases in this work contain the metal co-factor.Because the metalloenzymes are widely distributed and the electronic structures of metal ions are more complicated,the theoretical research about the metalloenzyme is attractive and difficult.This work is focused on solving some key questions involved in the enzymatic reaction,such as determine the possible binding modes of the substrates in the active sites,obtain the structures of the transition states and intermediates,clarify the optimal reaction pathway and analyze the effect of some key residues and ligands.This work can illuminate the catalytic mechanism at the atomistic level,which may contribute to understanding the biological function and the application of enzymes.The main contents in this work are as follow.(1)Mechanistic investigation of isonitrile formation catalyzed by ScoERecent structural and biochemical evidence showed that ScoE from S.coeruleorubidus is a non-heme iron/a-KG dependent decarboxylase,which catalyzes the formation of isonitrile group by desaturation and decarboxylation.This discovery offers an alternative mechanism for isonitrile formation.The other isonitrile synthases,such as IsnA,XnPvcA or AmbI1/AmbI2,convert R-CH(-NH2)-CO2-to R-CH(-N--C)-CO2-by introducing additional carbon unit,however,ScoE catalyzes the conversion of R-NH-CH2-CO2-to R-N-≡C through oxidative decarboxylation.To explore the catalytic mechanism of ScoE,on the basis of the high-resolution crystal structure,the enzyme-substrate complex models were constructed,and a series of combined QM/MM calculations were performed.Our results reveal that the ScoE-catalyzed reaction contains two decoupled desaturation and decarboxylation The FeⅣ-oxo-triggered desaturation includes two consecutive H-abstractions,which are similar to the C-C single bond desaturation catalyzed by other non-heme iron/a-KG dependent desaturases.In the second stage reaction,the decarboxylation of substrate radical generated by H-abstraction was calculated to be quite easy,whereas the previously proposed decarboxylation that involves the hydroxylated intermediate was calculated to be difficult.Importantly,the electron transfer from the substrate to the iron center is the key factor for lowering the barrier of decarboxylation.Thus,the central iron ion is not only responsible for H-abstraction,but also acts as an electron relay station for decarboxylation.In addition,this electron transfer was found to be coupled with a proton transfer,in which R310 and the associated H-bonding network play a critical role.In general,the first C-N desaturation is the rate-limiting step of the whole catalysis with an overall energy barrier of 17.6 or 16.9 kcal/mol in two competitive pathways,qualitatively agreeing with the estimated free energy(17.9-18.1 kcal/mol)from experiments.These results may provide useful information for understanding the biosynthesis of isonitrile and the oxidative decarboxylation catalyzed by non-heme iron/a-KG dependent enzymes.(2)Mechanistic study of two nogalamycin synthases SnoK and SnoNThe non-heme iron/a-ketoglutarate dependent enzymes SnoK and SnoN from Streptomyces nogalater are involved in the biosynthesis of anthracycline nogalamycin.Allhough they have similar active sites,SnoK is responsible for carbocyclization whereas SnoN solely catalyzes the hydroxyl epimerization.Herein,we performed docking,molecular simulations,and a series of combined quantum mechanics and molecular mechanics(QM/MM)calculations to illuminate the mechanisms of two enzymes.The catalytic reactions of two enzymes occur on the quintet state surface.For SnoK,the whole reaction includes two separated hydrogen-abstraction steps and one radical addition,and the latter step is calculated to be rate limiting with an energy barrier of 21.7 kcal/mol.Residue D106 is confirmed to participate in the construction of hydrogen bond network,which plays a crucial role in positioning the bulky substrate in a specific orientation.Moreover,it is found that SnoN is only responsible for the hydrogen-abstraction of the intermediate,and no residue was suggested to be suitable for donating a hydrogen atom to the substrate radical,which further confirms the suggestion based on experiments that either a cellular reductant or another enzyme protein could donate a hydrogen atom to the substrate.Our docking results coincide with the previous structural study that the different roles of two enzymes are achieved by minor changes in the alignment of the substrates in front of the reactive ferryl-oxo species.This work highlights the reaction mechanisms catalyzed by SnoK and SnoN,which is helpful for engineering the enzymes for the biosynthesis of anthracycline nogalamycin.(3)Theoretical insight into reaction mechanism of the streptozocin synthase SznFStreptozotocin is an N-nitrosourea natural product from bacterial,which are prominent carcinogens and important cancer chemotherapeutics.Recent experiments show the complete biosynthetic pathway of streptozotocin,in which SznF enzyme catalyzes an oxidative rearrangement of the guanidine group of Nω-methyl-L-arginine to generate an N-nitrosourea product.Although lots of work has been done about the structure and function of enzymes involved in the biosynthesis of streptozotocin,the catalytic mechanism of SznF enzyme is still uncertain.Based on the high-resolution crystal structure,the enzyme-O2-substrate complex models were constructed.Then,a series of combined QM/MM calculations were performed.Our results show that the substrate shows a bidentate binding to the iron center and the hexa-coordinate model of the center iron ion is obtained.The calculation results reveal two binding modes of substrate to the iron ion,which can convert each other.The spin density values show that electron transfer occurs from the substrate to dioxygen via the center iron ion,thus both substrate and dioxygen can be activated by binding to the center iron ion.The catalytic reaction occurs on the quintet state surface.A total of five steps are involved:Op abstracts the hydrogen atom from substrate,a cyclic peroxidation intermediate is formed by attacking the substrate,the formation of NO by breaking the Nε-Cε bond and the Op-Od bond,Fe-OH species abstracts the hydrogen atom from substrate,the substrate is attacked by NO to form an N-nitrosourea compound.The H-abstraction by Fe-OH is calculated to be rate limiting with an energy barrier of 21.0 kcal/mol.These results highlight the reaction mechanism catalyzed by SznF and provide useful information for understanding the biosynthesis of streptozotocin.(4)Insight into the catalytic mechanism of the quercetinase QueDFLAQuercetin 2,4-dioxygenase from Streptomyces sp.strain FLA(QueDFLA)is an enzyme of the monocupin family,which catalyzes the oxidative ring-cleaving reaction of quercetin using nickel ion as the active site cofactor,and the iron ion that is necessary for most dioxygenases only shows low reactivity.To understand the reaction mechanism and the activation of dioxygen by nickel ion,we performed QM/MM calculations to elucidate the reaction details and the special activation mechanism of this unique enzyme.Our calculations reveal two binding modes of dioxygen to the nickel ion,which can convert each other.Due to the overlap between the vacant d orbitals of nickel and the lone pair p orbitals of dioxygen and quercetin,electron transfer occurs from the quercetin to dioxygen via the nickel center,thus,both dioxygen and quercetin can be activated by their binding to the nickel ion.On the basis of our calculations,the triplet reactant complex favors the catalytic reaction,and the whole reaction contains four elementary steps.In particular,a nonchemical process,the Op-Od bond rotation along the nickel center,is suggested to be rate-limiting with free energy barrier of 19.9 kcal/mol.NBO analysis reveals that it the change of the coordination of Op with nickel ion that leads to the high energy barrier of this process.In general,owing to activation of substrate and dioxygen by nickel ion,the formation and collapse of the five-membered ring intermediate are quite easy,and the cleavage of O-O is in concert with the broken of two C-C bonds.Furthermore,when metal cofactor is replaced by iron ion,the rate-limiting step switches from the Op-Od bond rotation to the collapse of the five-membered ring intermediate,corresponding to free energy barrier of 30.3 kcal/mol.This study sheds insight into the reaction mechanism of QueD and contributes to our general understanding of other nickel containing enzymes.更多还原