D'Ambrosio Katia, Limauro Danila, Pedone Emilia, Galdi Ilaria, Pedone Carlo, Bartolucci Simonetta, De Simone Giuseppina
Istituto di Biostrutture e Bioimmagini-CNR, via Mezzocannone 16, 80134 Naples, Italy.
Proteins. 2009 Sep;76(4):995-1006. doi: 10.1002/prot.22408.
Bcps constitute a group of antioxidant enzymes, belonging to the Prx family, that are widely distributed in bacteria, plants, and fungi. These proteins can contain two conserved cysteines within the CXXXXC motif. Recent studies demonstrated that though the role of the first cysteine is well defined, being the catalytic peroxidatic cysteine in all the members of this protein family, data on the function of the second cysteine are controversial and require further investigation. In this article, we report on the functional and structural characterization of Bcp1, an archaeal Bcp isolated from Sulfolobus solfataricus, which presents two conserved cysteine residues at positions 45 and 50. Functional studies revealed that this enzyme performs the catalytic reaction using an atypical 2-Cys mechanism, where Cys45 is the peroxidatic and Cys50 is the resolving cysteine. The X-ray structure of the double mutant C45S/C50S, representative of the fully reduced enzyme state, was determined at a resolution of 2.15 A, showing a Trx fold similar to that of other Prxs. Superposition with a structural homologue in the oxidized state provided, for the first time, a detailed description of the structural rearrangement necessary for a member of the Bcp family to perform the catalytic reaction. From this structural analysis, it emerges that a significant conformational change from a fully folded, to a locally unfolded form is required to form the intramolecular disulfide bond upon oxidation, according to the proposed reaction mechanism. Two residues, namely Arg53 and Asp54, which could play a role in this rearrangement, were also identified.
细菌过氧化氢酶（Bcps）是一组抗氧化酶，属于过氧化物还原酶（Prx）家族，广泛分布于细菌、植物和真菌中。这些蛋白质在CXXXXC基序内可含有两个保守的半胱氨酸。最近的研究表明，虽然第一个半胱氨酸的作用已明确界定，即作为该蛋白质家族所有成员中的催化过氧化物半胱氨酸，但关于第二个半胱氨酸功能的数据存在争议，需要进一步研究。在本文中，我们报告了从嗜热栖热菌中分离出的古细菌Bcp1的功能和结构特征，该酶在第45和50位呈现两个保守的半胱氨酸残基。功能研究表明，这种酶使用非典型的2-半胱氨酸机制进行催化反应，其中Cys45是过氧化物半胱氨酸，Cys50是分解半胱氨酸。以2.15 Å的分辨率测定了代表完全还原酶状态的双突变体C45S/C50S的X射线结构，显示出与其他Prx相似的硫氧还蛋白折叠。与氧化态的结构同源物叠加，首次详细描述了Bcp家族成员进行催化反应所需的结构重排。从这一结构分析中可以看出，根据提出的反应机制，氧化时需要从完全折叠状态到局部未折叠状态发生显著的构象变化，以形成分子内二硫键。还确定了可能在这种重排中起作用的两个残基，即Arg53和Asp54。
