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科学家发现光驱酶的结构及其在生物燃料中的潜在应用 精选

已有 2562 次阅读 2021-4-27 10:44 |个人分类:新观察|系统分类:海外观察

科学家发现光驱酶的结构及其在生物燃料中的潜在应用

诸平

 F1.large.jpg

Elucidation of the FAP photocycle by combining spectroscopic, biochemical, crystallographic, and computational studies.

illu_FAP_LCLS web header crop_0.jpg

A study using SLAC’s LCLS X-ray laser captured how light drives a series of complex structural changes in an enzyme called FAP, which catalyzes the transformation of fatty acids into starting ingredients for solvents and fuels. This drawing captures the starting state of the catalytic reaction. The dark green background represents the protein’s molecular structure. The enzyme’s light-sensing part, called the FAD cofactor, is shown at center right with its three rings absorbing a photon coming from bottom left. A fatty acid at upper left awaits transformation. The amino acid shown at middle left plays an important role in the catalytic cycle, and the red dot near the center is a water molecule. (Damien Sorigué/Université Aix-Marseille)

美国能源部/SLAC国家加速器实验室DOE/SLAC National Accelerator Laboratory2021422日提供的消息,尽管许多生物捕获并响应太阳光,但酶(催化生化反应的蛋白质)很少受光驱动。SLAC国家加速器实验室一项新的研究捕获了一种称为脂肪酸光脱羧酶(fatty acid photodecarboxylase简称FAP的复杂结构变化的完整周期,该酶源自微观藻类,可将脂肪酸转化为溶剂和燃料的原料即烷烃或烯烃。

尽管许多生物捕获并响应太阳光,但酶(催化生化反应的蛋白质)却很少受到光的驱动。到目前为止,科学家们仅识别出3种类型的天然光酶(photoenzymes)。最新的发现于2017年的是脂肪酸光脱羧酶(fatty acid photodecarboxylase简称FAP)。它源自微观藻类,它使用蓝光催化脂肪和油中的脂肪酸转化为烷烃和烯烃。

格勒诺布尔·阿尔卑斯大学生物结构研究所(Institute of Biologie Structurale at the Universite Grenoble Alpes)的研究小组负责人马丁·韦克(Martin Weik)说:越来越多的实验室设想将FAP用于绿色化学应用,因为烷烃和烯烃是溶剂和燃料(包括汽油和喷气燃料)的重要成分。而且脂肪酸在脂肪酸光脱羧酶的作用之下,仅一步即可转化为烷烃或烯烃。

马丁·韦克是此项新研究的主要研究人员,也是通讯作者之一,该研究捕获了FAP对光的响应所引起的结构变化的复杂序列,即光周期,该周期驱动脂肪酸的转化。尽管研究人员先前提出了FAP光循环,但其基本机理尚不清楚。科学家们不知道脂肪酸要花多长时间才能失去羧基,羧基是碳氢化合物长链末端连接的化学基团,脱羧是形成烯烃或烷烃的关键步骤。

SLAC科学家合作,在美国能源部SLAC国家加速器实验室的直线加速器相干光源(LCLS)上进行的实验,帮助回答了许多这些悬而未决的问题。研究人员于202149日在《科学》(Science)杂志网站发表了他们的研究结果——D. Sorigué, K. Hadjidemetriou, S. Blangy, G. Gotthard, A. Bonvalet, N. Coquelle, P. Samire, A. Aleksandrov, L. Antonucci, A. Benachir, S. Boutet, M. Byrdin, M. Cammarata, S. Carbajo, S. Cuiné, R. B. Doak, L. Foucar, A. Gorel, M. Grünbein, E. Hartmann, R. Hienerwadel, M. Hilpert, M. Kloos, T. J. Lane, B. Légeret, P. Legrand, Y. Li-Beisson, S. L. Y. Moulin, D. Nurizzo, G. Peltier, G. Schirò, R. L. Shoeman, M. Sliwa, X. Solinas, B. Zhuang, T. R. M. Barends, J.-P. Colletier, M. Joffre, A. Royant, C. Berthomieu, M. Weik, T. Domratcheva, K. Brettel, M. H. Vos, I. Schlichting, P. Arnoux, P. Müller, F. Beisson. Mechanism and dynamics of fatty acid photodecarboxylaseScience, 09 Apr 2021; 372 (6538): eabd5687. DOI: 10.1126/science.abd5687.

参与此项研究的有来自法国艾克斯·马塞大学(Aix-Marseille University)、格勒诺布尔大学(Université Grenoble Alpes)、位于法国格勒诺布尔的欧洲同步辐射中心(European Synchrotron Radiation Facility)、巴黎理工学院(Institut Polytechnique de Paris)、劳厄·郎之万研究所(Institut Laue Langevin)、巴黎-萨克雷大学(Université Paris-Saclay)、雷恩第一大学(University of Rennes 1)、法国SOLEIL同步加速器(Synchrotron SOLEIL)、里尔大学(Univ. Lille);美国SLAC国家加速器实验室(SLAC National Accelerator Laboratory)、德国马克斯-普朗克医学应用研究所(Max-Planck-Institut für Medizinische Forschung)以及俄罗斯莫斯科国立大学(Lomonosov Moscow State University)的研究人员。

工具箱中的所有工具All the tools in a toolbox

为了理解像FAP这样的光敏酶(light-sensitive enzyme),科学家使用了许多不同的技术来研究在很宽的时间范围内发生的过程-因为光子吸收发生在飞秒(10-15 s)内,而分子水平上的生物响应却通常在千分之一秒(10-3 s)内发生。

马丁·韦克说:我们由法国艾克斯·马塞大学的弗雷德里克·贝松(Frederic Beisson)领导的国际跨学科研究小组使用了许多技术,包括光谱学、晶体学和计算方法。” “正是这些不同知识的融合,使我们得以初步了解这种独特的酶是如何随时间和空间变化的。

该联合小组首先在他们自己国家的实验室中使用光谱学方法研究了催化过程的复杂步骤,该方法研究了样品中原子的电子和几何结构,包括化学键合和电荷。光谱实验确定了酶伴随每个步骤的中间状态,测量了酶的寿命并提供了有关其化学性质的信息。这些结果激发了对LCLS超快速功能的需求。

接下来,使用LCLS X射线自由电子激光(X-ray free-electron laser简称XFEL),通过连续飞秒晶体学(serial femtosecond crystallography简称SFX)提供了催化过程的结构图。在这些实验中,用光学激光脉冲击中微小的FAP微晶射流以启动催化反应,然后通过非常短的超亮X射线脉冲来测量酶结构的最终变化。

通过整合成千上万的这些测量值(通过使用光脉冲和X射线脉冲之间的各种时间延迟获得的测量值),研究人员能够跟踪酶随时间的结构变化。他们还通过不使用光学激光进行探测来确定酶的静止状态的结构。

令人惊讶的是,研究人员发现,在静止状态下,该酶的光敏部分称为FAD辅因子(FAD cofactor),具有弯曲的形状。这个辅因子就像一个捕获光子的天线。它吸收蓝光并启动催化过程,马丁·韦克说,我们认为FAD辅因子的起点是平面的,因此这种弯曲的构想是出乎意料的。

FAD辅助因子的弯曲形状实际上是首先在欧洲同步加速器辐射设施的X射线晶体学中发现的,但科学家们怀疑这种弯曲是辐射损伤的产物,这是同步加速器光源收集的晶体学数据的一个普遍问题。马丁·韦克说,只有SFX实验才能确认这种不寻常的配置,因为它们具有在破坏样品之前捕获结构信息的独特能力。

他补充说:这些实验得到了计算的补充,没有莫斯科国立大学的塔蒂亚娜·多姆拉切特娃(Tatiana Domratcheva)进行的高级量子计算,我们就不会理解我们的实验结果。

下一步Next steps

尽管人们对FAP的照片循环有了更好的了解,但仍存在尚未解决的问题。例如,研究人员知道二氧化碳是在催化过程的特定步骤中的特定时间和位置形成的,但是他们对二氧化碳离开酶时的状态并不知晓。

马丁·韦克说:在将来的X射线自由电子激光(XFEL)工作中,我们希望识别产品的性质,并以较小的步长为其过程拍照,以便更精细地解析该过程。这对于基础研究很重要,但它也可以帮助科学家修改酶以完成特定应用的任务。上述介绍仅供参考,欲了解更多信息敬请注意浏览原文或者相关报道

Light makes light work of fatty acids

Photosynthetic organisms are notable for their ability to capture light energy and use it to power biosynthesis. Some algae have gone a step beyond photosynthesis and can use light to initiate enzymatic photodecarboxylation of fatty acids, producing long-chain hydrocarbons. To understand this transformation, Sorigué et al. brought to bear an array of structural, computational, and spectroscopic techniques and fully characterized the catalytic cycle of the enzyme. These experiments are consistent with a mechanism starting with electron transfer from the fatty acid to a photoexcited oxidized flavin cofactor. Decarboxylation yields an alkyl radical, which is then reduced by back electron transfer and protonation rather than hydrogen atom transfer. The wealth of experimental data explains how algae harness light energy to produce alka(e)nes and provides an appealing model system for understanding enzyme-catalyzed photochemistry more generally.

Science, this issue p. eabd5687

Structured Abstract

INTRODUCTION

Photoenzymes are rare biocatalysts driven by absorption of a photon at each catalytic cycle; they inspire development of artificial photoenzymes with valuable activities. Fatty acid photodecarboxylase (FAP) is a natural photoenzyme that has potential applications in the bio-based production of hydrocarbons, yet its mechanism is far from fully understood.

RATIONALE

To elucidate the mechanism of FAP, we studied the wild-type (WT) enzyme from Chlorella variabilis (CvFAP) and variants with altered active-site residues using a wealth of techniques, including static and time-resolved crystallography and spectroscopy, as well as biochemical and computational approaches.

RESULTS

A 1.8-Å-resolution CvFAP x-ray crystal structure revealed a dense hydrogen-bonding network positioning the fatty acid carboxyl group in the vicinity of the flavin adenine dinucleotide (FAD) cofactor. Structures solved from free electron laser and low-dose synchrotron x-ray crystal data further highlighted an unusual bent shape of the oxidized flavin chromophore, and showed that the bending angle (14°) did not change upon photon absorption (step 1) or throughout the photocycle. Calculations showed that bending substantially affected the energy levels of the flavin. Structural and spectroscopic analysis of WT and mutant proteins targeting two conserved active-site residues, R451 and C432, demonstrated that both residues were crucial for proper positioning of the substrate and water molecules and for oxidation of the fatty acid carboxylate by 1FAD* (~300 ps in WT FAP) to form FAD●– (step 2). Time-resolved infrared spectroscopy demonstrated that decarboxylation occured quasi-instantaneously upon this forward electron transfer, consistent with barrierless bond cleavage predicted by quantum chemistry calculations and with snapshots obtained by time-resolved crystallography. Transient absorption spectroscopy in H2O and D2O buffers indicated that back electron transfer from FAD●– was coupled to and limited by transfer of an exchangeable proton or hydrogen atom (step 3). Unexpectedly, concomitant with FAD●– reoxidation (to a red-shifted form FADRS) in 100 ns, most of the CO2 product was converted, most likely into bicarbonate (as inferred from FTIR spectra of the cryotrapped FADRS intermediate). Calculations indicated that this catalytic transformation involved an active-site water molecule. Cryo-Fourier transform infrared spectroscopy studies suggested that bicarbonate formation (step 4) was preceded by deprotonation of an arginine residue (step 3). At room temperature, the remaining CO2 left the protein in 1.5 μs (step 4ʹ). The observation of residual electron density close to C432 in electron density maps derived from time-resolved and cryocrystallography data suggests that this residue may play a role in stabilizing CO2 and/or bicarbonate. Three routes for alkane formation were identified by quantum chemistry calculations; the one shown in the figure is favored by the ensemble of experimental data.

CONCLUSION

We provide a detailed and comprehensive characterization of light-driven hydrocarbon formation by FAP, which uses a remarkably complex mechanism including unique catalytic steps. We anticipate that our results will help to expand the green chemistry toolkit.




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