Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase
Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase
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DOI：
10.1073/pnas.0610007104
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发表时间：
2007-01-09
影响因子：
11.1
通讯作者：
Tong, Liang
中科院分区：
文献类型：
--
作者：
Forouhar, Farhad;Anderson, J. L. Ross;Tong, Liang
色氨酸2，3-双加氧酶（TDO）和吲哚胺2，3-双加氧酶（IDO）构成含血红素酶的重要但相对知之甚少的家族。在这里，我们报告了广泛的结构和生化研究的野油菜黄单胞菌TOO和相关的蛋白质SO 4414希瓦氏菌oneidensis，包括在1.6埃分辨率的催化活性，亚铁形式的TOO在一个二元复合物与底物L-色氨酸的结构。色氨酸的羧酸和铵部分通过与酶和血红素的丙酸酯基团的静电和氢键相互作用来识别，从而定义了L-立体特异性。第二，可能是变构，L-Trp结合位点存在于四聚体界面。血红素-铁的第六配位位点是空的，提供了双氧结合位点，其也涉及与L-Trp的铵部分和甘氨酸残基的酰胺氮的相互作用。吲哚环被正确地定位以在C2和C3原子处氧化。活性位点仅在二元复合物中完全形成，生物化学实验证实了酶的这种诱导配合行为。活性位点在催化过程中完全没有水，这得到了我们的电化学研究的支持，表明底物结合后酶显着稳定。
Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) constitute an important, yet relatively poorly understood, family of heme-containing enzymes. Here, we report extensive structural and biochemical studies of the Xanthomonas campestris TOO and a related protein SO4414 from Shewanella oneidensis, including the structure at 1.6-angstrom resolution of the catalytically active, ferrous form of TOO in a binary complex with the substrate L-Trp. The carboxylate and ammonium moieties of tryptophan are recognized by electrostatic and hydrogen-bonding interactions with the enzyme and a propionate group of the heme, thus defining the L-stereospecificity. A second, possibly allosteric, L-Trp-binding site is present at the tetramer interface. The sixth coordination site of the heme-iron is vacant, providing a dioxygen-binding site that would also involve interactions with the ammonium moiety Of L-Trp and the amide nitrogen of a glycine residue. The indole ring is positioned correctly for oxygenation at the C2 and C3 atoms. The active site is fully formed only in the binary complex, and biochemical experiments confirm this induced-fit behavior of the enzyme. The active site is completely devoid of water during catalysis, which is supported by our electrochemical studies showing significant stabilization of the enzyme upon substrate binding.