The redox coenzymes NADH and NADPH are broadly required for energy metabolism, biosynthesis and detoxification. Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes, a holistic understanding of the regulation and compartmentalization of NADH- and NADPH-dependent pathways is lacking, partly because of a lack of tools with which to investigate these processes in living cells. We have previously reported the use of the naturally occurring Lactobacillus brevis H₂O-forming NADH oxidase (LbNOX) as a genetic tool for manipulation of the NAD⁺/NADH ratio in human cells. Here, we present triphosphopyridine nucleotide oxidase (TPNOX), a rationally designed and engineered mutant of LbNOX that is strictly specific to NADPH. We characterized the effects of TPNOX expression on cellular metabolism and used it in combination with LbNOX to show how the redox states of mitochondrial NADPH and NADH pools are connected.

中文翻译：

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一种用于在活细胞中操作 NADP+/NADPH 的基因编码工具

氧化还原辅酶 NADH 和 NADPH 是能量代谢、生物合成和解毒所必需的。尽管对利用这些辅酶的特定酶和途径有详细的了解，但缺乏对 NADH 和 NADPH 依赖性途径的调节和划分的整体理解，部分原因是缺乏研究活细胞中这些过程的工具。我们之前曾报道过使用天然存在的短乳杆菌H ₂ O 形成 NADH 氧化酶 ( Lb NOX) 作为遗传工具来控制人类细胞中的 NAD ⁺ /NADH 比率。在这里，我们介绍了三磷酸吡啶核苷酸氧化酶 (TPNOX)，这是一种经过合理设计和工程改造的Lb突变体严格特定于 NADPH 的 NOX。我们表征了 TPNOX 表达对细胞代谢的影响，并将其与Lb NOX 结合使用，以显示线粒体 NADPH 和 NADH 池的氧化还原状态如何连接。