Oliveira Marcos A, Guimarães Beatriz G, Cussiol José R R, Medrano Francisco J, Gozzo Fábio C, Netto Luis E S
Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo 05508-900, Brazil.
J Mol Biol. 2006 Jun 2;359(2):433-45. doi: 10.1016/j.jmb.2006.03.054. Epub 2006 Apr 7.
Organic hydroperoxide resistance proteins (Ohr) belong to a family of proteins that possess thiol-dependent peroxidase activity endowed by reactive cysteine residues able to reduce peroxides. The crystal structure of Ohr from Xylella fastidiosa in complex with polyethylene glycol, providing insights into enzyme-substrate interactions is described herein. In addition, crystallographic studies, molecular modeling and biochemical assays also indicated that peroxides derived from long chain fatty acids could be the biological substrates of Ohr. Because different oxidation states of the reactive cysteine were present in the Ohr structures from X. fastidiosa, Pseudomonas aeruginosa and Deinococcus radiodurans it was possible to envisage a set of snapshots along the coordinate of the enzyme-catalyzed reaction. The redox intermediates of X. fastidiosa Ohr observed in the crystals were further characterized in solution by electrospray ionization mass spectrometry and by biochemical approaches. In this study, the formation of an intramolecular disulfide bond and oxidative inactivation through the formation of a sulfonic acid derivative was unequivocally demonstrated for the first time. Because Ohr proteins are exclusively present in bacteria, they may represent promising targets for therapeutical drugs. In this regard, the structural and functional analyses of Ohr presented here might be very useful.
有机氢过氧化物抗性蛋白（Ohr）属于一类蛋白质，这类蛋白质具有由能够还原过氧化物的活性半胱氨酸残基赋予的硫醇依赖性过氧化物酶活性。本文描述了来自桑氏假单胞菌（Xylella fastidiosa）的Ohr与聚乙二醇复合物的晶体结构，该结构为酶 - 底物相互作用提供了见解。此外，晶体学研究、分子建模和生化分析还表明，长链脂肪酸衍生的过氧化物可能是Ohr的生物底物。由于在来自桑氏假单胞菌、铜绿假单胞菌（Pseudomonas aeruginosa）和耐辐射球菌（Deinococcus radiodurans）的Ohr结构中存在活性半胱氨酸的不同氧化态，因此有可能设想沿着酶催化反应坐标的一系列快照。通过电喷雾电离质谱和生化方法在溶液中进一步表征了在晶体中观察到的桑氏假单胞菌Ohr的氧化还原中间体。在本研究中，首次明确证明了分子内二硫键的形成以及通过磺酸衍生物的形成导致的氧化失活。由于Ohr蛋白仅存在于细菌中，它们可能是治疗药物的有前景的靶点。在这方面，本文介绍的Ohr的结构和功能分析可能非常有用。
