Kehrer Daniel, Ahmed Hatim, Brinkmann Henner, Siebers Bettina
Department of Biology and Geography, Institute of Biology, Microbiology I, Universität Duisburg-Essen, Universitätsstr, 5, 45117 Essen, Germany.
BMC Genomics. 2007 Aug 31;8:301. doi: 10.1186/1471-2164-8-301.
The presence of the branched Entner-Doudoroff (ED) pathway in two hyperthermophilic Crenarchaea, the anaerobe Thermoproteus tenax and the aerobe Sulfolobus solfataricus, was suggested. However, so far no enzymatic information of the non-phosphorylative ED branch and especially its key enzyme - glycerate kinase - was available. In the T. tenax genome, a gene homolog with similarity to putative hydroxypyruvate reductase/glycerate dehydrogenase and glycerate kinase was identified.
The encoding gene was expressed in E. coli in a recombinant form, the gene product purified and the glycerate kinase activity was confirmed by enzymatic studies. The enzyme was active as a monomer and catalyzed the ATP-dependent phosphorylation of D-glycerate forming exclusively 2-phosphoglycerate. The enzyme was specific for glycerate and highest activity was observed with ATP as phosphoryl donor and Mg2+ as divalent cation. ATP could be partially replaced by GTP, CTP, TTP and UTP. The enzyme showed high affinity for D-glycerate (Km 0.02 +/- 0.01 mM, Vmax of 5.05 +/- 0.52 U/mg protein) as well as ATP (Km of 0.03 +/- 0.01 mM, Vmax of 4.41 +/- 0.04 U/mg protein), although at higher glycerate concentrations, substrate inhibition was observed. Furthermore, the enzyme was inhibited by its product ADP via competitive inhibition. Data bank searches revealed that archaeal glycerate kinases are members of the MOFRL (multi-organism fragment with rich leucine) family, and homologs are found in all three domains of life.
A re-evaluation of available genome sequence information as well as biochemical and phylogenetic studies revealed the presence of the branched ED pathway as common route for sugar degradation in Archaea that utilize the ED pathway. Detailed analyses including phylogenetic studies demonstrate the presence of three distinct glycerate kinase classes in extant organisms that share no common origin. The affiliation of characterized glycerate kinases with the different enzyme classes as well as their physiological/cellular function reveals no association with particular pathways but a separate phylogenetic distribution. This work highlights the diversity and complexity of the central carbohydrate metabolism. The data also support a key function of the conversion of glycerate to 2- or 3-phosphoglycerate via glycerate kinase in funneling various substrates into the common EMP pathway for catabolic and anabolic purposes.
有人提出在两种嗜热泉古菌中存在分支型Entner-Doudoroff（ED）途径，这两种古菌分别是厌氧的嗜热栖热菌和需氧的嗜热栖硫菌。然而，到目前为止，关于非磷酸化ED分支的酶学信息，尤其是其关键酶——甘油酸激酶，尚无可用信息。在嗜热栖热菌基因组中，鉴定出一个与假定的羟基丙酮酸还原酶/甘油酸脱氢酶和甘油酸激酶具有相似性的基因同源物。
编码基因以重组形式在大肠杆菌中表达，对基因产物进行纯化，并通过酶学研究证实了甘油酸激酶活性。该酶以单体形式具有活性，催化D-甘油酸的ATP依赖性磷酸化反应，专一性地生成2-磷酸甘油酸。该酶对甘油酸具有特异性，以ATP作为磷酰供体、Mg2+作为二价阳离子时活性最高。ATP可部分被GTP、CTP、TTP和UTP替代。该酶对D-甘油酸具有高亲和力（Km为0.02±0.01 mM，Vmax为5.05±0.52 U/mg蛋白质）以及对ATP也具有高亲和力（Km为0.03±0.01 mM，Vmax为4.41±0.04 U/mg蛋白质），不过在较高甘油酸浓度下会观察到底物抑制现象。此外，该酶会被其产物ADP通过竞争性抑制作用所抑制。数据库搜索显示，古菌甘油酸激酶是MOFRL（富含亮氨酸的多生物体片段）家族的成员，并且在生命的所有三个域中都发现了同源物。
对现有基因组序列信息以及生化和系统发育研究的重新评估表明，分支型ED途径作为利用ED途径的古菌中糖降解的常见途径而存在。包括系统发育研究在内的详细分析表明，现存生物中存在三种不同的甘油酸激酶类别，它们没有共同的起源。已表征的甘油酸激酶与不同酶类的归属及其生理/细胞功能表明，它们与特定途径没有关联，而是具有独立的系统发育分布。这项工作突出了中心碳水化合物代谢的多样性和复杂性。数据还支持通过甘油酸激酶将甘油酸转化为2-或3-磷酸甘油酸在将各种底物导入共同的EMP途径以用于分解代谢和合成代谢目的方面的关键作用。
