Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif.

可溶性吡啶核苷酸转氢酶 (STH) 是参与必需辅因子 NAD(H) 和 NADP(H) 氧化还原稳态的黄素酶。它们催化两个烟酰胺辅助因子之间还原当量的可逆转移。来自大肠杆菌(SthA) 的可溶性转氢酶已在体内和体外应用中广泛使用，可引导还原当量进行需要 NADPH 的反应。然而，仍然缺乏对 SthA 功能的机制了解。在这项工作中，我们提出了 SthA 的生化特征，首次关注黄素酶与分子氧的反应性。我们报告了 SthA 的氧化酶活性，该活性在转氢过程中和在没有氧化烟酰胺辅助因子作为电子受体的情况下发生。我们发现该反应产生活性氧过氧化氢和超氧阴离子。此外，我们还探讨了保守的 CXXXXT 基序的进化意义，该基序将 STH 与相关的黄素蛋白二硫键还原酶家族（其中 CXXXXC 基序是保守的）区分开来。我们的突变分析表明，与具有突变基序的酶变体相比，SthA 中的半胱氨酸和苏氨酸组合可提高转氢偶联效率并减少活性氧释放。这些结果强调了与分子氧的反应性以及进化上保守的序列基序的重要性，从而扩展了我们对 SthA 机制的理解。