Aberrant regulation of metabolic kinases by altered redox homeostasis substantially contributes to aging and various diseases, such as diabetes. We found that the catalytic activity of a conserved family of fructosamine-3-kinases (FN3Ks), which are evolutionarily related to eukaryotic protein kinases, is regulated by redox-sensitive cysteine residues in the kinase domain. The crystal structure of the FN3K homolog from Arabidopsis thaliana revealed that it forms an unexpected strand-exchange dimer in which the ATP-binding P-loop and adjoining β strands are swapped between two chains in the dimer. This dimeric configuration is characterized by strained interchain disulfide bonds that stabilize the P-loop in an extended conformation. Mutational analysis and solution studies confirmed that the strained disulfides function as redox “switches” to reversibly regulate the activity and dimerization of FN3K. Human FN3K, which contains an equivalent P-loop Cys, was also redox sensitive, whereas ancestral bacterial FN3K homologs, which lack a P-loop Cys, were not. Furthermore, CRISPR-mediated knockout of FN3K in human liver cancer cells altered the abundance of redox metabolites, including an increase in glutathione. We propose that redox regulation evolved in FN3K homologs in response to changing cellular redox conditions. Our findings provide insights into the origin and evolution of redox regulation in the protein kinase superfamily and may open new avenues for targeting human FN3K in diabetic complications.

氧化还原稳态改变对代谢激酶的异常调节在很大程度上导致衰老和各种疾病，例如糖尿病。我们发现果糖胺-3-激酶（FN3K）保守家族的催化活性与真核蛋白激酶在进化上相关，并受到激酶结构域中氧化还原敏感的半胱氨酸残基的调节。来自拟南芥的 FN3K 同源物的晶体结构表明，它形成了一种意想不到的链交换二聚体，其中 ATP 结合 P 环和相邻的 β 链在二聚体的两条链之间交换。这种二聚体构型的特征是紧张的链间二硫键，可将 P 环稳定在延伸构象中。突变分析和溶液研究证实，应变二硫化物作为氧化还原“开关”，可逆地调节 FN3K 的活性和二聚化。人类 FN3K 含有等效的 P 环 Cys，也是氧化还原敏感的，而祖先细菌 FN3K 同源物缺乏 P 环 Cys，则不是。此外，CRISPR介导的人类肝癌细胞中FN3K的敲除改变了氧化还原代谢物的丰度，包括谷胱甘肽的增加。我们提出，FN3K 同系物中的氧化还原调节是为了响应细胞氧化还原条件的变化而进化的。我们的研究结果提供了对蛋白激酶超家族中氧化还原调节的起源和进化的见解，并可能为糖尿病并发症中靶向人类 FN3K 开辟新途径。