Krepinsky K, Plaumann M, Martin W, Schnarrenberger C
Institute of Biology, Free University of Berlin, Germany; Institute of Botany III, University of Düsseldorf, Germany.
Eur J Biochem. 2001 May;268(9):2678-86. doi: 10.1046/j.1432-1327.2001.02154.x.
Previous attempts to purify chloroplast 6-phosphogluconate dehydrogenase (cp6PGDH), a key enzyme of the oxidative pentose phosphate pathway, have been unsuccessful due to rapid activity loss. An efficient purification protocol was developed and the enzyme from spinach leaves was purified 1000-fold to apparent homogeneity with a specific activity of 60 U.mg-1. The enzyme is a homodimer with subunits of 50 kDa. Antibodies raised against the purified cp6PGDH detected a 53-kDa protein from a crude extract, indicating alterations during purification. Purified cp6PGDH was microsequenced and the corresponding spinach cDNA was cloned using PCR techniques and degenerate primers. The cDNA for cytosolic 6PGDH from spinach was cloned for comparison. Phylogenetic analysis in the context of available homologues from eukaryotes and eubacteria revealed that animal and fungal cytosolic 6PGDH sequences are more similar to their homologues from gamma-proteobacteria, whereas plant 6PGDH is more similar to its cyanobacterial homologues. The ancestral gene for higher plant 6PGDH was acquired from the antecedent of plastids through endosymbiosis and gene transfer to the nucleus. A subsequent gene duplication gave rise to higher plant cytosolic 6PGDH, which assumed the function of its pre-existing cytosolic homologue through endosymbiotic gene replacement. The protein phylogeny of both 6PGDH and of the first enzyme of the oxidative pentose phosphate pathway, glucose-6-phosphate dehydrogenase, indicate a surprisingly close relationship between the plant and Trypanosoma brucei lineages, suggesting that T. brucei (a relative of Euglena gracilis) may be secondarily nonphotosynthetic.
先前曾尝试纯化氧化戊糖磷酸途径的关键酶——叶绿体6-磷酸葡萄糖酸脱氢酶（cp6PGDH），但由于其活性迅速丧失，这些尝试均未成功。我们开发了一种高效的纯化方案，从菠菜叶中纯化出的该酶纯化了1000倍，达到了表观均一性，比活性为60 U·mg-1。该酶是一种由50 kDa亚基组成的同型二聚体。针对纯化的cp6PGDH产生的抗体在粗提物中检测到一种53 kDa的蛋白质，这表明在纯化过程中发生了变化。对纯化的cp6PGDH进行了微量测序，并使用PCR技术和简并引物克隆了相应的菠菜cDNA。为了进行比较，还克隆了菠菜胞质6PGDH的cDNA。在真核生物和真细菌现有同源物的背景下进行的系统发育分析表明，动物和真菌的胞质6PGDH序列与其来自γ-变形菌的同源物更相似，而植物6PGDH与其蓝细菌同源物更相似。高等植物6PGDH的祖先基因是通过内共生作用从质体的前身获得的，并转移到了细胞核中。随后的基因复制产生了高等植物胞质6PGDH，它通过内共生基因替代承担了其原有的胞质同源物的功能。6PGDH和氧化戊糖磷酸途径的第一种酶葡萄糖-6-磷酸脱氢酶的蛋白质系统发育表明，植物和布氏锥虫谱系之间存在惊人的密切关系，这表明布氏锥虫（纤细裸藻的近亲）可能是次生非光合的。
