Roles of distal aspartate and arginine of B-class dye-decolorizing peroxidase in heterolytic hydrogen peroxide cleavage
Roles of distal aspartate and arginine of B-class dye-decolorizing peroxidase in heterolytic hydrogen peroxide cleavage
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DOI：
10.1074/jbc.ra118.004773
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发表时间：
2018-09-21
影响因子：
4.8
通讯作者：
Obinger, Christian
中科院分区：
文献类型：
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作者：
Pfanzagl, Vera;Nys, Kevin;Obinger, Christian
染料脱色过氧化物酶（DyPs）是最近分类的依赖于过氧化氢的血红素过氧化物酶家族。虽然广泛分布有超过5000个注释基因，并因其生物技术潜力而受到欢迎，但其反应机制的详细生化表征仍然有限。在这里，我们提出了高分辨率晶体结构的WT B类DyP从致病性细菌肺炎克雷伯氏菌（KpDyP）（1.6埃）和变体D143 A（1.3埃），R232 A（1.9埃），和D143 A/R232 A（1.1埃）。我们证明了消除DyP典型的远端残基Asp-143和Arg-232对（i）光谱和氧化还原性质，（ii）过氧化氢异裂的动力学，（iii）低自旋氰化物络合物的形成，以及（iv）氧代铁（IV）卟啉π阳离子自由基（化合物I）的稳定性和反应性的影响。结构和功能研究表明，远端天冬氨酸负责H2 O2的去质子化和化合物I的不良氧化能力。远端精氨酸的消除促进远端血红素腔的塌陷，包括一个进入通道的阻断和催化天冬氨酸的构象变化。我们还提供了在KpDyP中形成氧代铁（IV）型化合物II的证据，其在418、527和553 nm处具有最大吸光度。综上所述，提出了B类DyPs的过氧化物酶循环的反应机制。我们的观察挑战的想法，对传统的芳香族底物的过氧化物酶活性相关的B类DyPs的生理作用。
Dye-decolorizing peroxidases (DyPs) represent the most recently classified hydrogen peroxide-dependent heme peroxidase family. Although widely distributed with more than 5000 annotated genes and hailed for their biotechnological potential, detailed biochemical characterization of their reaction mechanism remains limited. Here, we present the high-resolution crystal structures of WT B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) (1.6 angstrom) and the variants D143A (1.3 angstrom), R232A (1.9 angstrom), and D143A/R232A (1.1 angstrom). We demonstrate the impact of elimination of the DyP-typical, distal residues Asp-143 and Arg-232 on (i) the spectral and redox properties, (ii) the kinetics of heterolytic cleavage of hydrogen peroxide, (iii) the formation of the low-spin cyanide complex, and (iv) the stability and reactivity of an oxoiron(IV)porphyrin pi-cation radical (Compound I). Structural and functional studies reveal that the distal aspartate is responsible for deprotonation of H2O2 and for the poor oxidation capacity of Compound I. Elimination of the distal arginine promotes a collapse of the distal heme cavity, including blocking of one access channel and a conformational change of the catalytic aspartate. We also provide evidence of formation of an oxoiron(IV)-type Compound II in KpDyP with absorbance maxima at 418, 527, and 553 nm. In summary, a reaction mechanism of the peroxidase cycle of B-class DyPs is proposed. Our observations challenge the idea that peroxidase activity toward conventional aromatic substrates is related to the physiological roles of B-class DyPs.