Structural insights on the efficient catalysis of hydroperoxide reduction by Ohr: Crystallographic and molecular dynamics approaches

Organic hydroperoxide resistance (Ohr) enzymes are highly efficient Cys-based peroxidases that play central roles in bacterial response to fatty acid hydroperoxides and peroxynitrite, two oxidants that are generated during host-pathogen interactions. In the active site of Ohr proteins, the conserved Arg (Arg19 in Ohr from Xylella fastidiosa) and Glu (Glu51 in Ohr from Xylella fastidiosa) residues, among other factors, are involved in the extremely high reactivity of the peroxidatic Cys (Cp) toward hydroperoxides. In the closed state, the thiolate of Cp is in close proximity to the guanidinium group of Arg19. Ohr enzymes can also assume an open state, where the loop containing the catalytic Arg is far away from Cp and Glu51. Here, we aimed to gain insights into the putative structural switches of the Ohr catalytic cycle. First, we describe the crystal structure of Ohr from Xylella fastidiosa (XfOhr) in the open state that, together with the previously described XfOhr structure in the closed state, may represent two snapshots along the coordinate of the enzyme-catalyzed reaction. These two structures were used for the experimental validation of molecular dynamics (MD) simulations. MD simulations employing distinct protonation states and in silico mutagenesis indicated that the polar interactions of Arg19 with Glu51 and Cp contributed to the stabilization of XfOhr in the closed state. Indeed, Cp oxidation to the disulfide state facilitated the switching of the Arg19 loop from the closed to the open state. In addition to the Arg19 loop, other portions of XfOhr displayed high mobility, such as a loop rich in Gly residues. In summary, we obtained a high correlation between crystallographic data, MD simulations and biochemical/enzymatic assays. The dynamics of the Ohr enzymes are unique among the Cys-based peroxidases, in which the active site Arg undergoes structural switches throughout the catalytic cycle, while Cp remains relatively static.

有机氢过氧化物抗性酶（Organic hydroperoxide resistance, Ohr）是一类高效的半胱氨酸依赖型过氧化物酶，在细菌应对脂肪酸氢过氧化物与过氧亚硝酸盐的过程中发挥核心作用——这两类氧化剂均产生于宿主-病原体互作过程中。在Ohr蛋白的活性位点中，保守精氨酸残基（来自速生木杆菌Xylella fastidiosa的Ohr中为Arg19）与谷氨酸残基（同来源Ohr中为Glu51）等因素，共同决定了过氧化物酶活性半胱氨酸（Cp）对氢过氧化物的极高反应活性。当处于闭合构象时，Cp的硫醇盐与Arg19的胍基紧密相邻。Ohr酶还可呈现开放构象，此时包含催化性精氨酸的环区远离Cp与Glu51。本研究旨在深入解析Ohr催化循环中推测存在的结构转换机制。首先，我们解析了速生木杆菌来源Ohr（XfOhr）的开放构象晶体结构；结合此前已报道的闭合构象XfOhr结构，这两项成果可代表酶促反应坐标上的两个关键快照。我们利用这两种结构对分子动力学（MD）模拟进行了实验验证。通过采用不同质子化状态的体系与计算机诱变分析，结果显示Arg19与Glu51、Cp之间的极性相互作用，有助于XfOhr闭合构象的稳定。实际上，Cp被氧化为二硫键状态后，会促进Arg19所在环区从闭合构象向开放构象转换。除Arg19所在环区外，XfOhr的其他区域也表现出较高的流动性，例如一段富含甘氨酸残基的环区。综上，我们的晶体学数据、MD模拟结果与生化/酶学实验结果之间具有高度相关性。Ohr酶的动力学特征在半胱氨酸依赖型过氧化物酶中独具特色：其活性位点精氨酸在整个催化循环中发生结构转换，而Cp则保持相对静态。