PDF(Pubmed)
Abstract：
NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2\'s requirement for activation.
摘要：
NADPH氧化酶（NOX）是跨膜蛋白，在真核生物和原核生物中广泛传播，产生活性氧（ROS）。真核生物使用ROS产物在关键(病理)生理过程中进行先天免疫防御和信号传导。尽管最近人类NOX亚型的结构，电子转移的激活仍未被完全理解。SpNOX,来自肺炎链球菌的同源物，由于其本构活性，可以作为探索NOX家族中电子转移的强大模型。此处揭示了SpNOX全长和脱氢酶（DH）结构域构建体的晶体结构。分离的DH结构域充当黄素还原酶，并且两种构建体都使用NADPH或NADH作为底物。我们的发现表明，氢化物从NAD（P）H转移到FAD是电子转移中的限速步骤。我们确定了F397在烟酰胺获得黄素异咯嗪中的意义，并确认了DH和跨膜（TM）结构域的黄素结合贡献。与相关酶的比较表明，血红素的远端通路可能会影响最终的电子受体，虽然DH和TM的相对位置不一定与活性相关，与以前的建议相反。而是建议要求进行内部重组，在DH域内，从休息状态切换到活动状态。因此，SpNOX似乎是活跃的NOX2的良好模型，这使我们能够对NOX2的激活要求提出解释。
