Institute of Systems, Molecular and Integrative Biology, University of Liverpoolgrid.10025.36, Liverpool, United Kingdom.
Centre for Proteome Research, Institute of Integrative Biology, University of Liverpoolgrid.10025.36, Liverpool, United Kingdom.
mBio. 2022 Apr 26;13(2):e0362921. doi: 10.1128/mbio.03629-21. Epub 2022 Mar 28.
Carboxysomes are anabolic bacterial microcompartments that play an essential role in carbon fixation in cyanobacteria and some chemoautotrophs. This self-assembling organelle encapsulates the key CO-fixing enzymes, Rubisco, and carbonic anhydrase using a polyhedral protein shell that is constructed by hundreds of shell protein paralogs. The α-carboxysome from the chemoautotroph Halothiobacillus neapolitanus serves as a model system in fundamental studies and synthetic engineering of carboxysomes. In this study, we adopted a QconCAT-based quantitative mass spectrometry approach to determine the stoichiometric composition of native α-carboxysomes from H. neapolitanus. We further performed an in-depth comparison of the protein stoichiometry of native α-carboxysomes and their recombinant counterparts heterologously generated in Escherichia coli to evaluate the structural variability and remodeling of α-carboxysomes. Our results provide insight into the molecular principles that mediate carboxysome assembly, which may aid in rational design and reprogramming of carboxysomes in new contexts for biotechnological applications. A wide range of bacteria use special protein-based organelles, termed bacterial microcompartments, to encase enzymes and reactions to increase the efficiency of biological processes. As a model bacterial microcompartment, the carboxysome contains a protein shell filled with the primary carbon fixation enzyme Rubisco. The self-assembling organelle is generated by hundreds of proteins and plays important roles in converting carbon dioxide to sugar, a process known as carbon fixation. In this study, we uncovered the exact stoichiometry of all building components and the structural plasticity of the functional α-carboxysome, using newly developed quantitative mass spectrometry together with biochemistry, electron microscopy, and enzymatic assay. The study advances our understanding of the architecture and modularity of natural carboxysomes. The knowledge learned from natural carboxysomes will suggest feasible ways to produce functional carboxysomes in other hosts, such as crop plants, with the overwhelming goal of boosting cell metabolism and crop yields.
羧基体是一种合成代谢细菌微区室，在蓝细菌和一些化能自养生物的碳固定中发挥着重要作用。这种自组装细胞器利用多面体形蛋白质壳来封装关键的 CO 固定酶，Rubisco 和碳酸酐酶，该蛋白质壳由数百个壳蛋白同源物构建而成。来自化能自养菌 Halothiobacillus neapolitanus 的α-羧基体被用作基础研究和羧基体合成工程的模型系统。在这项研究中，我们采用基于 QconCAT 的定量质谱方法来确定来自 H. neapolitanus 的天然α-羧基体的化学计量组成。我们进一步对天然α-羧基体及其在大肠杆菌中异源产生的重组对应物的蛋白质化学计量进行了深入比较，以评估α-羧基体的结构可变性和重塑。我们的研究结果提供了对介导羧基体组装的分子原理的深入了解，这可能有助于在新的生物技术应用背景下对羧基体进行理性设计和重新编程。
许多细菌使用特殊的基于蛋白质的细胞器，称为细菌微区室，将酶和反应包裹起来，以提高生物过程的效率。作为一种模型细菌微区室，羧基体包含一个充满主要碳固定酶 Rubisco 的蛋白质壳。自组装细胞器由数百种蛋白质产生，在将二氧化碳转化为糖的过程中起着重要作用，这一过程称为碳固定。在这项研究中，我们使用新开发的定量质谱法以及生物化学、电子显微镜和酶测定法，揭示了所有构建成分的精确化学计量和功能α-羧基体的结构可塑性。该研究增进了我们对天然羧基体的结构和模块化的理解。从天然羧基体中获得的知识将为在其他宿主（如作物植物）中产生功能性羧基体提供可行的方法，其主要目标是提高细胞代谢和作物产量。
