Evolutionary conserved light regulation of Calvin cycle activity by NADPH-mediated reversible phosphoribulokinase/CP12/ glyceraldehyde-3-phosphate dehydrogenase complex dissociation

For higher plant chloroplasts, two key enzymes of the Calvin cycle, phosphoribulokinase (EC 2.7.1.19) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.13), have recently been shown to be oligomerized onto the nonenzymatic peptide CP12. Enzymatic activity depends on complex dissociation, mediated by NADPH. The discovery of genes for CP12 in mosses, green algae, and cyanobacteria, together with the analysis of equivalent multiprotein complexes of Chlamydomonas and Synechocystis suggests that light regulation of Calvin cycle activity via NADPH-mediated reversible phosphoribulokinase/CP12/GAPDH complex dissociation is conserved in all photosynthetic organisms, prokaryotes and eukaryotes. In vitro complex reconstitution assays with heterologously expressed Synechocystis wild-type and mutagenized CP12 demonstrate a conserved subunit composition, stoichiometry, and topology in this complex. Further finding of genes, coding for chimeric proteins, carrying CP12 or parts of it as genetic fusions, indicates that evolution has used the peptide loops of CP12 as universal modules to keep various enzymatic activities under the control of NADP(H). These fusion events occurred at least twice in evolution. First was the fusion of the duplicated genes for CP12 and the ORF4 protein of Anabaena variabilis to the chimeric gene for the heterocyst-specific expressed ORF3 protein, most probably involved in N(2) fixation. A second gene fusion, which led to the higher plant chloroplast-specific GAPDH subunit, GAPB, has taken place during the transition from water- to land plants.

针对高等植物叶绿体，卡尔文循环的两种关键酶——磷酸核酮糖激酶（phosphoribulokinase，EC 2.7.1.19）与甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase，GAPDH，EC 1.2.1.13）——近期被证实可寡聚结合至无酶活性肽CP12。其酶活性依赖于烟酰胺腺嘌呤二核苷酸磷酸还原型（NADPH）介导的复合物解离。在苔藓、绿藻与蓝细菌中鉴定到CP12的编码基因，结合对衣藻与集胞藻等效多蛋白复合物的分析，提示通过NADPH介导的可逆磷酸核酮糖激酶/CP12/GAPDH复合物解离来调控卡尔文循环活性的机制，在所有光合生物（涵盖原核与真核生物）中均保守存在。利用异源表达的集胞藻野生型与诱变型CP12开展的体外复合物重构实验证实，该复合物的亚基组成、化学计量比与空间拓扑结构均具有保守性。进一步发现的编码嵌合蛋白的基因（其中CP12或其部分片段作为遗传融合元件存在）表明，进化过程中曾将CP12的肽环作为通用模块，使多种酶活性受NADP(H)的调控。这类基因融合事件在进化历程中至少发生过两次：第一次为多变鱼腥藻的CP12重复基因与ORF4蛋白编码基因发生融合，形成异形胞特异性表达的ORF3蛋白编码嵌合基因，该蛋白极可能参与氮气固定过程；第二次基因融合事件发生于水生植物向陆生植物的演化过渡阶段，最终造就了高等植物叶绿体特异性的GAPDH亚基GAPB。