Biochemical and biophysical properties of a highly active recombinant arginase from Leishmania (Leishmania) amazonensis and subcellular localization of native enzyme
Biochemical and biophysical properties of a highly active recombinant arginase from Leishmania (Leishmania) amazonensis and subcellular localization of native enzyme
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DOI：
10.1016/j.molbiopara.2008.02.011
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发表时间：
2008-06-01
影响因子：
1.5
通讯作者：
Floeter-Winter, Lucile Maria
中科院分区：
文献类型：
--
作者：
da Silva, Edson Roberto;Laranjeira da Silva, Maria Fernanda;Floeter-Winter, Lucile Maria
精氨酸酶（L-精氨酸脒基水解酶，E. C. 3.5.3.1）是催化L-精氨酸水解为L-鸟氨酸和尿素的金属酶。在利什曼原虫属中，该酶的生物学作用可能涉及在巨噬细胞感染时调节NO的产生。在此之前，我们克隆并鉴定了利什曼原虫亚马逊亚种（Leishmania amazonensis）的β-淀粉酶基因。在本研究中，我们成功地在大肠杆菌中表达了重组酶。大肠杆菌，并进行了天然和重组酶的生物化学和生物物理特性。我们得到了K-M和V-max。对于天然酶，分别为23.9（+/-0.96）mM和192.3 μ mol/min mg蛋白（+/-14.3）。对于重组对应物，K-M为21.5（+/- 0.90）mM，V-max为144.9（+/- 8.9）μ mol/min mg。针对重组蛋白的抗体证实了前鞭毛体中酶的糖体细胞定位。来自光散射和小角X射线散射的数据表明，三聚体状态是蛋白质的活性形式。我们凭经验确定在室温下锰洗涤是纯化活性酶的最佳条件。重组蛋白与固定化镍的相互作用也使我们能够确认组氨酸在位置3和324的结构处置。所确定的结构参数提供了大量的数据，以促进寻找选择性抑制剂的寄生虫来源的利什曼病的治疗，这可能随后指向一个候选人。(c)2008 Elsevier B. V.保留所有权利。
Arginase (L-arginine amidinohydrolase, E.C. 3.5.3.1) is a metalloenzyme that catalyses the hydrolysis Of L-arginine to L-ornithine and urea. In Leishmania spp., the biological role of the enzyme may be involved in modulating NO production upon macrophage infection. Previously, we cloned and characterized the arginase gene from Leishmania (Leishmania) amazonensis. In the present work, we successfully expressed the recombinant enzyme in E. coli and performed biochemical and biophysical characterization of both the native and recombinant enzymes. We obtained K-M and V-max. values of 23.9(+/- 0.96) mM and 192.3 mu mol/min mg protein (+/- 14.3), respectively, for the native enzyme. For the recombinant counterpart, K-M was 21.5(+/- 0.90) mM and V-max was 144.9(+/- 8.9) mu mol/min mg. Antibody against the recombinant protein confirmed a glycosomal cellular localization of the enzyme in promastigotes. Data from light scattering and small angle X-ray scattering showed that a trimeric state is the active form of the protein. We determined empirically that a manganese wash at room temperature is the best condition to purify active enzyme. The interaction of the recombinant protein with the immobilized nickel also allowed us to confirm the structural disposition of histidine at positions 3 and 324. The determined structural parameters provide substantial data to facilitate the search for selective inhibitors of parasitic sources of arginase, which could subsequently point to a candidate for leishmaniasis therapy. (c) 2008 Elsevier B.V. All rights reserved.