Evolutionary optimization of enzymes for their operation in the C4 photosynthetic pathway: the case of NADP-malic enzyme

C4 光合作用途径酶的进化优化：以 NADP-苹果酸酶为例

• 批准号: 441941117
• 负责人: Professorin Dr. Veronica Maurino
• 金额: --
• 依托单位: Abteilung Molekulare Pflanzenphysiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/441941117?language=en
• 关键词: Evolutionary; optimization; enzymes; their; operation

The ancestors of C4 plants evolved a biochemical pump to concentrate CO2 at the site of Rubisco, leading to greater photosynthetic efficiency. The evolutionary adaptation of cellular activities to the C4 syndrome was based on the molecular adaptation of existing enzymes, in most cases involving gene duplications followed by neo-functionalization, accompanied by changes in the regulation and biochemical properties of the gene products.A central step in C4 photosynthesis is the generation of high CO2 concentrations through the decarboxylation of a C4 acid in the bundle sheath cell (BSC) chloroplasts. Most agronomically important C4 plants, including maize, sorghum, and sugar cane, belong to the NADP-malic enzyme (ME) subtype of C4, which predominantly uses NADP-ME for this purpose. During the night, when the photosynthetic pathway is inactive, the enzymatic activity of the C4-specific isoform of NADP-ME is regulated through two processes: on the one hand, the enzyme partially loses its active quaternary oligomerization state; on the other hand, it is inhibited by malate. These regulatory mechanisms are important for the efficient concentration of CO2 in BSC chloroplasts. We recently identified specific amino acids important for the molecular adaptions of C4-NADP-ME based on strict differential conservation of amino acids, combined with solving the crystal structures of maize and sorghum C4-NADP-ME and the biochemical analysis of enzyme mutants. Several of these amino acid substitutions likely evolved to implement the necessary regulatory adaptions. In this project, we aim to elucidate the molecular mechanisms underlying the evolution of the two major regulatory properties of C4-NADP-ME, and to analyze these regulatory processes in vivo. We focus on the single C4 lineage Andropogoneae within the Panicoideae subfamily of the Poaceae, which includes the C4 grasses maize and sorghum. We will combine crystallization analyses with in silico modelling to analyze how specific amino acids present only in the C4-NADP-ME isoform influence the structural and kinetic properties of the enzyme. Through biochemical analysis and small-angle-X-ray-scattering measurements of N-terminal mutants of NADP-ME, we will determine which other amino acids are involved in the changes and stabilization of the oligomeric states. We will establish the underlying molecular mechanism of malate inhibition through the biochemical analysis of specific enzyme mutants. Furthermore, we will identify other amino acids involved in the adaptation of C4-NADP-ME in the Andropogoneae by producing and analyzing further recombinant NADP-ME mutants. Finally, to evaluate these regulatory processes in vivo, we will introduce specific mutations into the gene coding for C4-NADP-ME in maize via the CRISPR-Cas9 technology and will perform phenotypic, biochemical, and physiological analyses of the obtained plant lines.

C4 植物的祖先进化出了一种生化泵，将二氧化碳集中在 Rubisco 位置，从而提高光合作用效率。细胞活动对 C4 综合征的进化适应是基于现有酶的分子适应，在大多数情况下涉及基因复制，然后进行新功能化，并伴随着基因产物的调节和生化特性的变化。C4 光合作用的一个核心步骤是通过束鞘细胞中的 C4 酸脱羧产生高浓度的 CO2 （BSC）叶绿体。大多数农艺上重要的 C4 植物，包括玉米、高粱和甘蔗，都属于 C4 的 NADP-苹果酸酶 (ME) 亚型，该亚型主要使用 NADP-ME 来实现这一目的。在夜间，当光合途径不活跃时，NADP-ME的C4特异性亚型的酶活性通过两个过程进行调节：一方面，酶部分失去其活性的四级寡聚状态；另一方面，它受到苹果酸的抑制。这些调节机制对于 BSC 叶绿体中 CO2 的有效浓缩非常重要。我们最近基于氨基酸的严格差异保守性，结合解决玉米和高粱C4-NADP-ME的晶体结构以及酶突变体的生化分析，确定了对C4-NADP-ME分子适应重要的特定氨基酸。其中一些氨基酸取代可能是为了实施必要的监管调整而进化的。在这个项目中，我们的目标是阐明 C4-NADP-ME 两个主要调控特性进化的分子机制，并分析这些体内调控过程。我们重点关注禾本科黍亚科中的单个 C4 谱系 Androgogonae，其中包括 C4 禾本科植物玉米和高粱。我们将结晶分析与计算机建模相结合，分析仅存在于 C4-NADP-ME 亚型中的特定氨基酸如何影响酶的结构和动力学特性。通过对 NADP-ME N 末端突变体的生化分析和小角 X 射线散射测量，我们将确定哪些其他氨基酸参与了寡聚状态的变化和稳定。我们将通过特定酶突变体的生化分析来建立苹果酸抑制的潜在分子机制。此外，我们将通过产生和分析进一步的重组 NADP-ME 突变体来鉴定与 C4-NADP-ME 在仙人掌亚目中的适应有关的其他氨基酸。最后，为了在体内评估这些调控过程，我们将通过 CRISPR-Cas9 技术在玉米中编码 C4-NADP-ME 的基因中引入特定突变，并对获得的植物品系进行表型、生化和生理分析。