College of Life Science, Nankai University, Tianjin, China 300071.
FASEB J. 2011 Feb;25(2):653-64. doi: 10.1096/fj.10-170811. Epub 2010 Nov 3.
Human protoporphyrinogen IX oxidase (hPPO), a mitochondrial inner membrane protein, converts protoporphyrinogen IX to protoporphyrin IX in the heme biosynthetic pathway. Mutations in the hPPO gene cause the inherited human disease variegate porphyria (VP). In this study, we report the crystal structure of hPPO in complex with the coenzyme flavin adenine dinucleotide (FAD) and the inhibitor acifluorfen at a resolution of 1.9 Å. The structural and biochemical analyses revealed the molecular details of FAD and acifluorfen binding to hPPO as well as the interactions of the substrate with hPPO. Structural analysis and gel chromatography indicated that hPPO is a monomer rather than a homodimer in vitro. The founder-effect mutation R59W in VP patients is most likely caused by a severe electrostatic hindrance in the hydrophilic binding pocket involving the bulky, hydrophobic indolyl ring of the tryptophan. Forty-seven VP-causing mutations were purified by chromatography and kinetically characterized in vitro. The effect of each mutation was demonstrated in the high-resolution crystal structure.
人原卟啉原 IX 氧化酶（hPPO）是一种线粒体内膜蛋白，在血红素生物合成途径中可将原卟啉原 IX 转化为原卟啉 IX。hPPO 基因的突变会导致遗传性人类疾病斑驳卟啉症（VP）。在本研究中，我们报道了 hPPO 与辅酶黄素腺嘌呤二核苷酸（FAD）和抑制剂氟磺胺草醚复合物的晶体结构，分辨率为 1.9Å。结构和生化分析揭示了 FAD 和氟磺胺草醚与 hPPO 结合的分子细节，以及底物与 hPPO 的相互作用。结构分析和凝胶层析表明，hPPO 在体外是单体而不是同源二聚体。VP 患者中的 founder-effect 突变 R59W 很可能是由于亲水结合口袋中涉及大体积疏水性吲哚环的色氨酸的强烈静电阻碍所致。通过层析法纯化了 47 种导致 VP 的突变体，并在体外进行了动力学特征分析。每个突变的影响都在高分辨率晶体结构中得到了证明。
