人源原卟啉原氧化酶结构与功能的研究
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摘要
原卟啉原氧化酶(protoporphyrinogen oxidase,PPO, EC1.3.3.4)是叶绿素和血红素共同合成途径中的最后一个酶。广泛存在于生物界中。可催化无色的原卟啉原Ⅸ氧化,失去六个质子生成褐色的光敏物质原卟啉Ⅸ。
在生物体中,原卟啉原氧化酶活性的降低,会导致底物原卟啉原Ⅸ的积累,泄露到胞质中后,会自发氧化生成原卟啉Ⅸ,在光照的条件下,原卟啉Ⅸ激发产生高活性的氧自由基,破坏脂质、蛋白质等重要的生物大分子结构,最终导致细胞死亡。
在人体中,原卟啉原氧化酶基因的突变会导致一种显性遗传病——混合型卟啉病。在植物中,原卟啉原氧化酶活性的抑制可使细胞致死,是新型除草剂的作用靶标。因此原卟啉原氧化酶在医学上和农学上都有重要的研究价值。
通过大量实验,解析了1.9A高分辨率的人源原卟啉原氧化酶的晶体结构,并从以下几方面进行了分析研究：
一、具体分析了人源原卟啉原氧化酶与辅酶FAD,抑制剂AF的相互作用；利用化学对接的方法拟合了原卟啉原氧化酶与底物的结合模式,并通过酶动力学研究验证了模型的合理性；
二、通过结构学分析以及生化实验数据,首次证实hPPO在溶液中以单体形式存在,而非过去人们认为的二聚体；
三、首次将全球范围内引起混合型卟啉病的44个点突变的突变体蛋白分别进行了体外克隆、表达、纯化及活性测定,并在高分辨率的结构上进行了致病机理的分析。
血红素的合成途径涉及到8种酶,其中后7种酶的缺损都会导致不同类型的卟啉病,这些酶中除了原卟啉原氧化酶外,结构都得到了解析。本论文对原卟啉原氧化酶结构的解析,完善了血红素合成途径中酶结构学的研究,不仅能为血红素的分子合成机制研究提供新思路,也为混合型卟啉病的预防与治疗提供新视角。
Protoporphyrinogen IX oxidase (PPO; EC1.3.3.4), is the last common enzyme for the transformation of protoporphyrinogen IX to protoporphyrin IX, and plays an important part in the biosynthesis of heme and chlorophyll.
The inhibition of PPO leads to the accumulation of protoporphyrinogen IX, which is then exported to the cytoplasm and oxidized to protoporphyrin IX spontaneously by oxygen. In the presence of light, the photosensitizing protoporphyrin IX can further generate singlet oxygen, which induces rapid lipid peroxidation and cell death. These special characteristics of PPO enzymes underlie their role in an inherited human disease called variegated prophyria (VP) and also make it an excellent target for herbicides.
In this study, we report the crystal structure of Human protoporphyrinogen IX oxidase (hPPO) in complex with the coenzyme flavin adenine dinucleotide (FAD) and the inhibitor acifluorfen at a resolution of1.9A.
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.
The human heme biosynthesis pathway consists of8enzymes. A deficiency in each of the last7enzymes corresponds with a variant of porphyria. The crystal structure of each these enzymes has been determined, except for hPPO. Here, with the current hPPO structure, we have all7structures at hand. We believe that these structures will provide important information not only for understanding the heme biosynthesis but also for further elucidating the molecular mechanism of porphyria.
在生物体中,原卟啉原氧化酶活性的降低,会导致底物原卟啉原Ⅸ的积累,泄露到胞质中后,会自发氧化生成原卟啉Ⅸ,在光照的条件下,原卟啉Ⅸ激发产生高活性的氧自由基,破坏脂质、蛋白质等重要的生物大分子结构,最终导致细胞死亡。
在人体中,原卟啉原氧化酶基因的突变会导致一种显性遗传病——混合型卟啉病。在植物中,原卟啉原氧化酶活性的抑制可使细胞致死,是新型除草剂的作用靶标。因此原卟啉原氧化酶在医学上和农学上都有重要的研究价值。
通过大量实验,解析了1.9A高分辨率的人源原卟啉原氧化酶的晶体结构,并从以下几方面进行了分析研究：
一、具体分析了人源原卟啉原氧化酶与辅酶FAD,抑制剂AF的相互作用；利用化学对接的方法拟合了原卟啉原氧化酶与底物的结合模式,并通过酶动力学研究验证了模型的合理性；
二、通过结构学分析以及生化实验数据,首次证实hPPO在溶液中以单体形式存在,而非过去人们认为的二聚体；
三、首次将全球范围内引起混合型卟啉病的44个点突变的突变体蛋白分别进行了体外克隆、表达、纯化及活性测定,并在高分辨率的结构上进行了致病机理的分析。
血红素的合成途径涉及到8种酶,其中后7种酶的缺损都会导致不同类型的卟啉病,这些酶中除了原卟啉原氧化酶外,结构都得到了解析。本论文对原卟啉原氧化酶结构的解析,完善了血红素合成途径中酶结构学的研究,不仅能为血红素的分子合成机制研究提供新思路,也为混合型卟啉病的预防与治疗提供新视角。
Protoporphyrinogen IX oxidase (PPO; EC1.3.3.4), is the last common enzyme for the transformation of protoporphyrinogen IX to protoporphyrin IX, and plays an important part in the biosynthesis of heme and chlorophyll.
The inhibition of PPO leads to the accumulation of protoporphyrinogen IX, which is then exported to the cytoplasm and oxidized to protoporphyrin IX spontaneously by oxygen. In the presence of light, the photosensitizing protoporphyrin IX can further generate singlet oxygen, which induces rapid lipid peroxidation and cell death. These special characteristics of PPO enzymes underlie their role in an inherited human disease called variegated prophyria (VP) and also make it an excellent target for herbicides.
In this study, we report the crystal structure of Human protoporphyrinogen IX oxidase (hPPO) in complex with the coenzyme flavin adenine dinucleotide (FAD) and the inhibitor acifluorfen at a resolution of1.9A.
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.
The human heme biosynthesis pathway consists of8enzymes. A deficiency in each of the last7enzymes corresponds with a variant of porphyria. The crystal structure of each these enzymes has been determined, except for hPPO. Here, with the current hPPO structure, we have all7structures at hand. We believe that these structures will provide important information not only for understanding the heme biosynthesis but also for further elucidating the molecular mechanism of porphyria.
引文
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[2]Poulson, R., The regulation of heme biosynthesis. Ann Clin Res,1976.8 Suppl 17:p.56-63.
[3]Matringe, M., J.M. Camadro, M.A. Block, et al., Localization within chloroplasts of protoporphyrinogen oxidase, the target enzyme for diphenylether-like herbicides. J Biol Chem,1992.267(7):p.4646-51.
[4]Panek, H. and M.R. O'Brian, A whole genome view of prokaryotic haem biosynthesis. Microbiology,2002.148(Pt 8):p.2273-82.
[5]Heinemann, I.U., M. Jahn and D. Jahn, The biochemistry of heme biosynthesis. Arch Biochem Biophys,2008.474(2):p.238-51.
[6]Ajioka, R.S., J.D. Phillips and J.P. Kushner, Biosynthesis of heme in mammals. Biochim Biophys Acta,2006.1763(7):p.723-36.
[7]Che, F.S., N. Watanabe, M. Iwano, et al., Molecular characterization and subcellular localization of protoporphyrinogen oxidase in spinach chloroplasts. Plant Physiol,2000. 124(1):p.59-70.
[8]Watanabe, N., F.S. Che, M. Iwano, et al., Dual targeting of spinach protoporphyrinogen oxidase II to mitochondria and chloroplasts by alternative use of two in-frame initiation codons. J Biol Chem,2001.276(23):p.20474-81.
[9]Poulson, R. and W.J. Polglase, The enzymic conversion of protoporphyrinogen IX to protoporphyrin Ⅸ. Protoporphyrinogen oxidase activity in mitochondrial extracts of Saccharomyces cerevisiae. J Biol Chem,1975.250(4):p.1269-74.
[10]Poulson, R., The enzymic conversion of protoporphyrinogen IX to protoporphyrin Ⅸ in mammalian mitochondria. J Biol Chem,1976.251(12):p.3730-3.
[11]Sun, L., X. Wen, Y. Tan, et al., Site-directed mutagenesis and computational study of the Y366 active site in Bacillus subtilis protoporphyrinogen oxidase. Amino Acids,2009.37(3): p.523-30.
[12]Qin, X., L. Sun, X. Wen, et al., Structural insight into unique properties of protoporphyrinogen oxidase from Bacillus subtilis. J Struct Biol,2010.170(1):p.76-82.
[13]Klemm, D.J. and L.L. Barton, Purification and properties of protoporphyrinogen oxidase from an anaerobic bacterium, Desulfovibrio gigas. J Bacteriol,1987.169(11):p.5209-15.
[14]Corradi, H.R., A.V. Corrigall, E. Boix, et al., Crystal structure of protoporphyrinogen oxidase from Myxococcus xanthus and its complex with the inhibitor acifluorfen. J Biol Chem,2006.281(50):p.38625-33.
[15]Dailey, H.A. and S.W. Karr, Purification and characterization of murine protoporphyrinogen oxidase. Biochemistry,1987.26(10):p.2697-701.
[16]Watanabe, N., F.S. Che, K. Terashima, et al., Purification and properties of protoporphyrinogen oxidase from spinach chloroplasts. Plant Cell Physiol,2000.41(7):p. 889-92.
[17]Koch, M., C. Breithaupt, R. Kiefersauer, et al., Crystal structure of protoporphyrinogen Ⅸ oxidase:a key enzyme in haem and chlorophyll biosynthesis. EMBO J,2004.23(8):p. 1720-8.
[18]Dailey, T.A. and H.A. Dailey, Identification of an FAD superfamily containing protoporphyrinogen oxidases, monoamine oxidases, and phytoene desaturase. Expression and characterization of phytoene desaturase of Myxococcus xanthus. J Biol Chem,1998. 273(22):p.13658-62.
[19]Wierenga, R.K., P. Terpstra and W.G. Hol, Prediction of the occurrence of the ADP-binding beta alpha beta-fold in proteins, using an amino acid sequence fingerprint. J Mol Biol,1986. 187(1):p.101-7.
[20]Dailey, T.A., H.A. Dailey, P. Meissner, et al., Cloning, sequence, and expression of mouse protoporphyrinogen oxidase. Arch Biochem Biophys,1995.324(2):p.379-84.
[21]Jacobs, J.M. and N.J. Jacobs, Oxidation of protoporphyrinogen to protoporphyrin, a step in chlorophyll and haem biosynthesis. Purification and partial characterization of the enzyme from barley organelles. Biochem J,1987.244(1):p.219-24.
[22]Corrigall, A.V., K.B. Siziba, M.H. Maneli, et al., Purification of and kinetic studies on a cloned protoporphyrinogen oxidase from the aerobic bacterium Bacillus subtilis. Arch Biochem Biophys,1998.358(2):p.251-6.
[23]Jacobs, N.J. and J.M. Jacobs, Fumarate as alternate electron acceptor for the late steps of anaerobic heme synthesis in Escherichia coli. Biochem Biophys Res Commun,1975.65(1): p.435-41.
[24]Jacobs, N.J. and J.M. Jacobs, Nitrate, fumarate, and oxygen as electron acceptors for a late step in microbial heme synthesis. Biochim Biophys Acta,1976.449(1):p.1-9.
[25]Sasarman, A., J. Letowski, G. Czaika, et al., Nucleotide sequence of the hemG gene involved in the protoporphyrinogen oxidase activity of Escherichia coli K.12. Can J Microbiol,1993. 39(12):p.1155-61.
[26]Sano, S. and S. Granick, Mitochondrial coproporphyrinogen oxidase and protoporphyrin formation. J Biol Chem,1961.236:p.1173-80.
[27]Akhtar, M., M.M. Abboud, G. Barnard, et al., Mechanism and stereochemistry of enzymic reactions involved in porphyrin biosynthesis. Philos Trans R Soc Lond B Biol Sci,1976. 273(924):p.117-36.
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