Cereijo Antonela E, Asencion Diez Matías D, Dávila Costa José S, Alvarez Héctor M, Iglesias Alberto A
Laboratorio de Enzimología Molecular, Instituto de Agrobiotecnología del Litoral, CONICET, Centro Científico Tecnológico, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral Santa Fe, Argentina.
Centro Regional de Investigación y Desarrollo Científico Tecnológico, Facultad de Ciencias Naturales Universidad Nacional de la Patagonia San Juan Bosco Comodoro Rivadavia, Argentina.
Front Microbiol. 2016 Jun 2;7:830. doi: 10.3389/fmicb.2016.00830. eCollection 2016.
Rhodococcus spp. are oleaginous bacteria that accumulate glycogen during exponential growth. Despite the importance of these microorganisms in biotechnology, little is known about the regulation of carbon and energy storage, mainly the relationship between glycogen and triacylglycerols metabolisms. Herein, we report the molecular cloning and heterologous expression of the gene coding for ADP-glucose pyrophosphorylase (EC 2.7.7.27) of Rhodococcus jostii, strain RHA1. The recombinant enzyme was purified to electrophoretic homogeneity to accurately characterize its oligomeric, kinetic, and regulatory properties. The R. jostii ADP-glucose pyrophosphorylase is a homotetramer of 190 kDa exhibiting low basal activity to catalyze synthesis of ADP-glucose, which is markedly influenced by different allosteric effectors. Glucose-6P, mannose-6P, fructose-6P, ribose-5P, and phosphoenolpyruvate were major activators; whereas, NADPH and 6P-gluconate behaved as main inhibitors of the enzyme. The combination of glucose-6P and other effectors (activators or inhibitors) showed a cross-talk effect suggesting that the different metabolites could orchestrate a fine regulation of ADP-glucose pyrophosphorylase in R. jostii. The enzyme exhibited some degree of affinity toward ATP, GTP, CTP, and other sugar-1P substrates. Remarkably, the use of glucosamine-1P was sensitive to allosteric activation. The relevance of the fine regulation of R. jostii ADP-glucose pyrophosphorylase is further analyzed in the framework of proteomic studies already determined for the bacterium. Results support a critical role for glycogen as a temporal reserve that provides a pool of carbon able of be re-routed to produce long-term storage of lipids under certain conditions.
红球菌属是一类产油细菌，在指数生长期积累糖原。尽管这些微生物在生物技术中具有重要意义，但关于碳和能量储存的调控，尤其是糖原与三酰甘油代谢之间的关系，人们了解甚少。在此，我们报道了约氏红球菌RHA1菌株中编码ADP - 葡萄糖焦磷酸化酶（EC 2.7.7.27）的基因的分子克隆和异源表达。将重组酶纯化至电泳纯，以准确表征其寡聚、动力学和调控特性。约氏红球菌ADP - 葡萄糖焦磷酸化酶是一种190 kDa的同四聚体，催化ADP - 葡萄糖合成的基础活性较低，其活性受到不同变构效应物的显著影响。葡萄糖 - 6 - 磷酸、甘露糖 - 6 - 磷酸、果糖 - 6 - 磷酸、核糖 - 5 - 磷酸和磷酸烯醇丙酮酸是主要激活剂；而NADPH和6 - 磷酸葡萄糖酸则是该酶的主要抑制剂。葡萄糖 - 6 - 磷酸与其他效应物（激活剂或抑制剂）的组合显示出串扰效应，表明不同代谢物可能协同对约氏红球菌中的ADP - 葡萄糖焦磷酸化酶进行精细调控。该酶对ATP、GTP、CTP和其他糖 - 1 - 磷酸底物表现出一定程度的亲和力。值得注意的是，氨基葡萄糖 - 1 - 磷酸的使用对变构激活敏感。在已针对该细菌进行的蛋白质组学研究框架内，进一步分析了约氏红球菌ADP - 葡萄糖焦磷酸化酶精细调控的相关性。结果支持糖原作为一种临时储备发挥关键作用，在某些条件下它能提供碳源，可重新导向用于产生脂质的长期储存。
