Soares Jose Sergio M, Gentile Agustina, Scorsato Valeria, Lima Aline da C, Kiyota Eduardo, Dos Santos Marcelo Leite, Piattoni Claudia V, Huber Steven C, Aparicio Ricardo, Menossi Marcelo
From the Departamento de Genética, Evolução e Bioagentes, Instituto de Biologia, Universidade Estadual de Campinas, Rua Monteiro Lobato, 255, C.P. 6109, Campinas, SP, Brazil.
the Laboratório de Biologia Estrutural e Cristalografia, Instituto de Química, Universidade Estadual de Campinas, C.P. 6154, Campinas, SP, Brazil.
J Biol Chem. 2014 Nov 28;289(48):33364-77. doi: 10.1074/jbc.M114.590125. Epub 2014 Oct 15.
Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H2O2 strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.
甘蔗是一种单子叶植物，其茎中蔗糖积累量可达干重的50%。目前对甘蔗中蔗糖积累所涉及的机制了解甚少，对于控制茎中蔗糖储存程度的因素也知之甚少。尿苷二磷酸葡萄糖焦磷酸化酶（UGPase；EC 2.7.7.9）是一种产生尿苷二磷酸葡萄糖的酶，尿苷二磷酸葡萄糖是蔗糖代谢和细胞壁生物合成的关键前体。这项工作的目的是深入了解ScUGPase-1的表达模式和控制蛋白质活性的调节机制。在发育过程中，ScUGPase-1的表达与节间的蔗糖含量呈负相关，在蔗糖含量不同的两个品种之间，仅观察到表达模式的细微差异。ScUGPase-1的细胞内定位表明，这种可溶性蛋白在叶片和节间均部分与膜结合。使用磷酸特异性抗体，我们观察到ScUGPase-1在体内叶片的可溶性和膜部分中Ser-419位点被磷酸化，但在节间未被磷酸化。对纯化的重组酶进行了尿苷二磷酸葡萄糖形成方向的动力学表征，酶活性受氧化还原修饰的影响。用H2O2预孵育强烈抑制了这种活性，而DTT可以使其逆转。小角X射线散射分析表明，二聚体界面位于C末端，并提供了溶液中甘蔗UGPase二聚体的第一个结构模型。
