Department of Biochemistry, University of Western Ontario, London, Ontario, Canada.
Protein Sci. 2024 Dec;33(12):e5234. doi: 10.1002/pro.5234.
Quinone reductases 1 and 2 (NQO1 and NQO2) are paralogous FAD-linked enzymes found in all amniotes. NQO1 and NQO2 have similar structures, and both catalyze the reduction of quinones and other electrophiles; however, the two enzymes differ in their cosubstrate preference. While NQO1 can use both redox couples NADH and NADPH, NQO2 is almost inactive with these cosubstrates and instead must use dihydronicotinamide riboside (NRH) and small synthetic cosubstrates such as N-benzyl-dihydronicotinamide (BNAH) for efficient catalysis. We used ancestral sequence reconstruction to investigate the catalytic properties of a predicted common ancestor and two additional ancestors from each of the evolutionary pathways to extant NQO1 and NQO2. In all cases, the small nicotinamide cosubstrates NRH and BNAH were good cosubstrates for the common ancestor and the enzymes along both the NQO1 and NQO2 lineages. In contrast, with NADH as cosubstrate, extant NQO1 evolved to a catalytic efficiency 100 times higher than the common ancestor, while NQO2 has evolved to a catalytic efficiency 3000 times lower than the common ancestor. The evolutionary analysis combined with site-directed mutagenesis revealed a potential site of interaction for the ADP portion of NAD(P)H in NQO1 that is altered in charge and structure in NQO2. The results indicate that while NQO1 evolved to have greater efficiency with NAD(P)H, befitting an enzymatic function in cells, NQO2 was under selective pressure to acquire extremely low catalytic efficiency with NAD(P)H. These divergent trajectories have implications for the functions of both enzymes.
醌还原酶 1 和 2（NQO1 和 NQO2）是在所有羊膜动物中发现的具有相似结构的 FAD 连接酶。NQO1 和 NQO2 具有相似的结构，都能催化醌类和其他亲电试剂的还原；然而，两种酶在辅助因子偏好上存在差异。虽然 NQO1 可以使用 NADH 和 NADPH 这两种氧化还原对，但 NQO2 几乎不能利用这些辅助因子，而是必须使用二氢烟酰胺核糖（NRH）和小的合成辅助因子，如 N-苄基二氢烟酰胺（BNAH），才能有效地催化。我们使用祖先序列重建来研究预测的共同祖先和每个进化途径到现存的 NQO1 和 NQO2 的两个额外祖先的催化特性。在所有情况下，小的烟酰胺辅助因子 NRH 和 BNAH 都是共同祖先和 NQO1 和 NQO2 两个谱系中的酶的良好辅助因子。相比之下，以 NADH 作为辅助因子，现存的 NQO1 进化出比共同祖先高 100 倍的催化效率，而 NQO2 则进化出比共同祖先低 3000 倍的催化效率。进化分析结合定点突变揭示了 NAD(P)H 的 ADP 部分在 NQO1 中的一个潜在相互作用位点，该位点在 NQO2 中电荷和结构发生了改变。结果表明，虽然 NQO1 进化出更高的 NAD(P)H 催化效率，适合细胞中的酶功能，但 NQO2 受到选择压力，以获得极低的 NAD(P)H 催化效率。这些不同的轨迹对两种酶的功能都有影响。
