Zeida Ari, Reyes Aníbal M, Lichtig Pablo, Hugo Martín, Vazquez Diego S, Santos Javier, González Flecha F Luis, Radi Rafael, Estrin Dario A, Trujillo Madia
Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República , Montevideo 11800, Uruguay.
Biochemistry. 2015 Dec 15;54(49):7237-47. doi: 10.1021/acs.biochem.5b00758. Epub 2015 Nov 30.
Peroxiredoxins (Prxs) constitute a ubiquitous family of Cys-dependent peroxidases that play essential roles in reducing hydrogen peroxide, peroxynitrite, and organic hydroperoxides in almost all organisms. Members of the Prx subfamilies show differential oxidizing substrate specificities that await explanations at a molecular level. Among them, alkyl hydroperoxide reductases E (AhpE) is a novel subfamily comprising Mycobacterium tuberculosis AhpE and AhpE-like proteins expressed in some bacteria and archaea. We previously reported that MtAhpE reacts ∼10(4) times faster with an arachidonic acid derived hydroperoxide than with hydrogen peroxide, and suggested that this surprisingly high reactivity was related to the presence of a hydrophobic groove at the dimer interface evidenced in the crystallography structure of the enzyme. In this contribution we experimentally confirmed the existence of an exposed hydrophobic patch in MtAhpE. We found that fatty acid hydroperoxide reduction by the enzyme showed positive activation entropy that importantly contributed to catalysis. Computational dynamics indicated that interactions of fatty acid-derived hydroperoxides with the enzyme properly accommodated them inside the active site and modifies enzyme's dynamics. The computed reaction free energy profile obtained via QM/MM simulations is consistent with a greater reactivity in comparison with hydrogen peroxide. This study represents new insights on the understanding of the molecular basis that determines oxidizing substrate selectivity in the peroxiredoxin family, which has not been investigated at an atomic level so far.
过氧化物酶（Prxs）是一类广泛存在的依赖半胱氨酸的过氧化物酶家族，在几乎所有生物体中，它们在还原过氧化氢、过氧亚硝酸盐和有机氢过氧化物方面发挥着重要作用。Prx亚家族的成员表现出不同的氧化底物特异性，这有待于在分子水平上进行解释。其中，烷基氢过氧化物还原酶E（AhpE）是一个新的亚家族，包括结核分枝杆菌AhpE以及在一些细菌和古细菌中表达的AhpE样蛋白。我们之前报道过，结核分枝杆菌AhpE与花生四烯酸衍生的氢过氧化物反应的速度比与过氧化氢反应的速度快约10⁴倍，并提出这种惊人的高反应性与该酶晶体结构中在二聚体界面处存在的疏水凹槽有关。在本论文中，我们通过实验证实了结核分枝杆菌AhpE中存在一个暴露的疏水区域。我们发现该酶对脂肪酸氢过氧化物的还原表现出正的活化熵，这对催化作用有重要贡献。计算动力学表明，脂肪酸衍生的氢过氧化物与该酶的相互作用使其能够恰当地容纳在活性位点内，并改变了酶的动力学特性。通过量子力学/分子力学（QM/MM）模拟获得的计算反应自由能分布与过氧化氢相比具有更高的反应活性。这项研究为理解决定过氧化物酶家族氧化底物选择性分子基础提供了新的见解，到目前为止尚未在原子水平上对此进行研究。
