Departamento de Química Inorgánica, Analítica y Química Física/INQUIMAE-CONICET Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, Buenos Aires C1428EHA, Argentina.
Biochemistry. 2011 Dec 20;50(50):10910-8. doi: 10.1021/bi201439m. Epub 2011 Nov 23.
Tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) are the only two heme proteins that catalyze the oxidation reaction of tryptophan (Trp) to N-formylkynurenine. While human IDO is able to oxidize both L- and D-Trp, human TDO (hTDO) displays major specificity for L-Trp. In this work, we aim to interrogate the molecular basis for the substrate stereoselectivity of hTDO. Our previous molecular dynamics simulation studies of Xanthomonas campestris TDO (xcTDO) showed that a hydrogen bond between T254 (T342 in hTDO) and the ammonium group of the substrate is present in the L-Trp-bound enzyme, but not in the D-Trp-bound enzyme. The fact that this is the only notable structural alteration induced by the change in the stereo structure of the substrate prompted us to produce and characterize the T342A mutant of hTDO to evaluate the structural role of T342 in controlling the substrate stereoselectivity of the enzyme. The experimental results indicate that the mutation only slightly perturbs the global structural properties of the enzyme but totally abolishes the substrate stereoselectivity. Molecular dynamics simulations of xcTDO show that T254 controls the substrate stereoselectivity of the enzyme by (i) modulating the hydrogen bonding interaction between the NH(3)(+) group and epoxide oxygen of the ferryl-indole 2,3-epoxide intermediate of the enzyme and (ii) regulating the dynamics of two active site loops, loop(250-260) and loop(117-130), critical for substrate binding.
色氨酸双加氧酶（TDO）和吲哚胺 2,3-双加氧酶（IDO）是唯二能够催化色氨酸（Trp）氧化生成 N-甲酰犬尿氨酸的血红素蛋白。虽然人类 IDO 能够氧化 L-和 D-Trp，但人类 TDO（hTDO）对 L-Trp 表现出主要的特异性。在这项工作中，我们旨在探究 hTDO 底物立体选择性的分子基础。我们之前对黄单胞菌 TDO（xcTDO）的分子动力学模拟研究表明，T254（hTDO 中的 T342）与底物的铵基之间存在氢键，而在 D-Trp 结合的酶中则不存在。这是由于底物立体结构的变化引起的唯一显著结构改变，这促使我们产生并表征 hTDO 的 T342A 突变体，以评估 T342 在控制酶的底物立体选择性中的结构作用。实验结果表明，突变仅略微干扰酶的整体结构性质，但完全消除了底物的立体选择性。xcTDO 的分子动力学模拟表明，T254 通过以下两种方式控制酶的底物立体选择性：（i）调节酶的 ferryl-吲哚 2,3-环氧化物中间物中 NH3(+) 基团与环氧化物氧之间的氢键相互作用；（ii）调节两个关键的活性位点环，环（250-260）和环（117-130）的动力学，这些环对底物结合至关重要。
