Departamento de Bioquímica, Facultad de Química, Conjunto E., Universidad Nacional Autónoma de México, CDMX, Mexico.
Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mexico.
BMC Plant Biol. 2019 Jan 15;19(1):27. doi: 10.1186/s12870-018-1605-x.
Seed germination is a crucial process in the plant life cycle when a dramatic variation of type and sugar content occurs just as the seed is hydrated. The production of hexose 6 phosphate is a key node in different pathways that are required for a successful germination. Hexokinase (HXK) is the only plant enzyme that phosphorylates glucose (Glc), so it is key to fueling several metabolic pathways depending on their substrate specificity, metabolite regulatory responses and subcellular localization. In maize, the HXK family is composed of nine genes, but only six of them (ZmHXK4-9) putatively encode catalytically active enzymes. Here, we cloned and functionally characterized putative catalytic enzymes to analyze their metabolic contribution during germination process.
From the six HXKs analyzed here, only ZmHXK9 has minimal hexose phosphorylating activity even though enzymatic function of all isoforms (ZmHXK4-9) was confirmed using a yeast complementation approach. The kinetic parameters of recombinant proteins showed that ZmHXK4-7 have high catalytic efficiency for Glc, fructose (Fru) and mannose (Man), ZmHXK7 has a lower Km for ATP, and together with ZmHXK8 they have lower sensitivity to inhibition by ADP, G6P and N-acetylglucosamine than ZmHXK4-6 and ZmHXK9. Additionally, we demonstrated that ZmHXK4-6 and ZmHXK9 are located in the mitochondria and their location relies on the first 30 amino acids of the N-terminal domain. Otherwise, ZmHXK7-8 are constitutively located in the cytosol. HXK activity was detected in cytosolic and mitochondrial fractions and high Glc and Fru phosphorylating activities were found in imbibed embryos.
Considering the biochemical characteristics, location and the expression of ZmHXK4 at onset of germination, we suggest that it is the main contributor to mitochondrial activity at early germination times, at 24 h other ZmHXKs also contribute to the total activity. While in the cytosol, ZmHXK7 could be responsible for the activity at the onset of germination, although later, ZmHXK8 also contributes to the total HXK activity. Our observations suggest that the HXKs may be redundant proteins with specific roles depending on carbon and ATP availability, metabolic needs, or sensor requirements. Further investigation is necessary to understand their specific or redundant physiological roles.
种子萌发是植物生命周期中的一个关键过程，在此过程中，种子在水合时会发生类型和糖含量的剧烈变化。己糖 6 磷酸的产生是成功萌发所需的不同途径的关键节点。己糖激酶（HXK）是唯一能够磷酸化葡萄糖（Glc）的植物酶，因此它是为几种代谢途径提供燃料的关键，具体取决于其底物特异性、代谢物调节反应和亚细胞定位。在玉米中，HXK 家族由 9 个基因组成，但其中只有 6 个（ZmHXK4-9）推测编码具有催化活性的酶。在这里，我们克隆并功能表征了推定的催化酶，以分析它们在萌发过程中的代谢贡献。
在分析的 6 个 HXK 中，只有 ZmHXK9 具有最小的己糖磷酸化活性，尽管使用酵母互补方法证实了所有同工型（ZmHXK4-9）的酶功能。重组蛋白的动力学参数表明，ZmHXK4-7 对 Glc、果糖（Fru）和甘露糖（Man）具有高催化效率，ZmHXK7 对 ATP 的 Km 较低，与 ZmHXK8 一起，它们对 ADP、G6P 和 N-乙酰葡萄糖胺的抑制作用比对 ZmHXK4-6 和 ZmHXK9 敏感。此外，我们证明 ZmHXK4-6 和 ZmHXK9 位于线粒体中，它们的位置依赖于 N 端结构域的前 30 个氨基酸。否则，ZmHXK7-8 位于细胞质中。在胞质和线粒体部分检测到 HXK 活性，在吸胀的胚胎中发现高 Glc 和 Fru 磷酸化活性。
考虑到生化特性、定位和 ZmHXK4 在萌发开始时的表达，我们认为它是早期萌发时线粒体活性的主要贡献者，在 24 小时时其他 ZmHXK 也有助于总活性。在细胞质中，ZmHXK7 可能负责萌发开始时的活性，尽管后来 ZmHXK8 也有助于总 HXK 活性。我们的观察表明，HXK 可能是具有特定作用的冗余蛋白，具体作用取决于碳和 ATP 的可用性、代谢需求或传感器需求。需要进一步研究以了解它们的特定或冗余的生理作用。
