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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基因编码的工具，用于在活细胞中操纵NADP + / NADPH

氧化还原辅酶NADH和NADPH广泛用于能量代谢，生物合成和排毒。尽管对特定酶和利用这些辅酶的途径有详细的了解，但对NADH和NADPH依赖性途径的调节和区室化缺乏全面的了解，部分原因是缺乏研究活细胞中这些过程的工具。我们以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）作为操纵人细胞中NAD ⁺ / NADH比的遗传工具。在这里，我们介绍三磷酸吡啶核苷酸氧化酶（TPNOX），这是一种合理设计和改造的Lb突变体严格针对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库的氧化还原状态如何连接。