Nolan Katie, Usai Remigio, Li Bingnan, Jordan Stephanie, Wang Yifan
Department of Chemistry, University of Georgia, Athens, Georgia 30602, United States.
ACS Catal. 2025 Jun 3;15(12):10391-10404. doi: 10.1021/acscatal.5c01932. eCollection 2025 Jun 20.
RufO is a unique cytochrome P450 enzyme (CYP) involved in the biosynthesis of rufomycin, an antituberculosis cyclic peptide featuring an unusual nitrated tyrosine. Recent studies have clarified RufO's role in producing ribosomally synthesized and post-translationally modified peptides (RiPPs). Despite growing interest in nitrating enzymes and RiPP biosynthesis, the mechanism by which RufO recognizes and nitrates its pentapeptide substrate, MRYLH, remains poorly understood. In this study, we use a combination of spectroscopic, kinetic, and structural techniques to elucidate the molecular basis for peptide binding and heme-based nitration in RufO. Peptide binding is an endothermic process with a dissociation constant of 0.78 μM. Unlike most CYPs, RufO does not undergo the typical spin state conversion nor exhibit a significant increase in reduction potential upon substrate binding. The minimal perturbation to the heme center may lead to RufO's lack of specificity for redox partners. However, significant shifts in the vibrational frequencies of carbonyl complexes upon substrate binding indicate a more polar heme distal site that favors a nonlinear binding conformation of diatomic gas molecules. These distinctive features contrast with TxtE, the only other CYP known to catalyze aromatic nitration. A 1.51 Å resolution crystal structure reveals that substrate binding induces significant conformational changes in the distal pocket, particularly in the regions interacting with Arg-2 and His-5 of the MRYLH peptide. While Tyr-3 is positioned similarly to its counterpart in P450, a paralog that catalyzes peptide cross-linking, an extended hydrogen-bonding network constraining His-5 is unique to RufO and likely contributes to its distinct nitration activity. Furthermore, transient kinetic data suggest the sequential binding of O followed by NO and characterize a ferric-superoxo intermediate essential for the nitration activity. This study provides valuable insights into the substrate specificity and catalytic mechanisms of CYPs involved in nitration reactions and RiPP biosynthesis.
鲁夫霉素氧化酶（RufO）是一种独特的细胞色素P450酶（CYP），参与鲁夫霉素的生物合成，鲁夫霉素是一种具有不寻常硝化酪氨酸的抗结核环肽。最近的研究阐明了RufO在产生核糖体合成和翻译后修饰肽（RiPPs）中的作用。尽管人们对硝化酶和RiPP生物合成的兴趣日益增加，但RufO识别并硝化其五肽底物MRYLH的机制仍知之甚少。在本研究中，我们结合光谱、动力学和结构技术来阐明RufO中肽结合和基于血红素的硝化作用的分子基础。肽结合是一个吸热过程，解离常数为0.78 μM。与大多数CYP不同，RufO不会经历典型的自旋态转换，也不会在底物结合后表现出还原电位的显著增加。对血红素中心的最小扰动可能导致RufO对氧化还原伙伴缺乏特异性。然而，底物结合后羰基配合物振动频率的显著变化表明血红素远端位点更具极性，有利于双原子气体分子的非线性结合构象。这些独特的特征与TxtE形成对比，TxtE是已知的另一种催化芳香族硝化的CYP。1.51 Å分辨率的晶体结构表明，底物结合会在远端口袋中引起显著的构象变化，特别是在与MRYLH肽的Arg-2和His-5相互作用的区域。虽然Tyr-3的位置与其在催化肽交联的旁系同源物P450中的对应物相似，但限制His-5的扩展氢键网络是RufO独有的，可能有助于其独特的硝化活性。此外，瞬态动力学数据表明O先于NO顺序结合，并表征了硝化活性所必需的铁-超氧中间体。本研究为参与硝化反应和RiPP生物合成的CYP的底物特异性和催化机制提供了有价值的见解。
