A sorghum ascorbate peroxidase with four binding sites has activity against ascorbate and phenylpropanoids
A sorghum ascorbate peroxidase with four binding sites has activity against ascorbate and phenylpropanoids
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具有四个结合位点的高粱抗坏血酸过氧化物酶具有抗坏血酸和苯丙素的活性
DOI：
10.1093/plphys/kiac604
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发表时间：
2022
期刊：
影响因子：
7.4
通讯作者：
Kang, ChulHee
中科院分区：
文献类型：
--
作者：
Zhang, Bixia;Lewis, Jacob A;Vermerris, Wilfred;Sattler, Scott E;Kang, ChulHee
在植物中，过氧化氢是作为酶反应和防御反应的副产物产生的。抗坏血酸过氧化物酶(APX)是清除细胞毒性过氧化氢的关键酶。本文报道了高粱胞质APX(Sobic.001G410200)的晶体结构。虽然SbAPX的整体结构与其他APX相似，但SbAPX独特地显示了四个结合的抗坏血酸，而不是一个。除了在其他APX中发现的ɣ-血红素口袋外，抗坏血酸还结合在δ-MESO和两个暴露在溶剂中的口袋上。与多个结合位点的存在相一致，我们的结果表明依赖于H_2O_2的抗坏血酸氧化表现出正的协同性。在两个表面位置结合的抗坏血酸建立了一个复杂的质子网络，而抗坏血酸在ɣ-血红素边缘和δ-中位。根据晶体结构、稳态动力学和定点突变结果，ɣ-血红素边缘和表面的抗坏血酸分子都有望参与氧化反应。我们提供的证据表明，APX依赖于H_2O_2氧化抗坏血酸在ɣ-血红素边缘产生C2-水合双环半酮形式的脱氢抗坏血酸，表明单一结合的抗坏血酸有两次连续的电子转移。此外，δ-Meso位与几种有机化合物共享，包括对香豆酸和其他苯丙烷类化合物，用于潜在的自由基反应。ɣ-血红素边缘的关键残基(R172a)的定点突变仅部分降低了聚合活性。因此，APX用抗坏血酸去除应激产生的过氧化氢，并利用同样的过氧化氢通过苯丙烷类化合物的氧化聚合来潜在地加固细胞壁，以响应应激。
In planta, H2O2is produced as a by-product of enzymatic reactions and during defense responses. Ascorbate peroxidase (APX) is a key enzyme involved in scavenging cytotoxic H2O2. Here, we report the crystal structure of cytosolic APX from sorghum (Sorghum bicolor) (Sobic.001G410200). While the overall structure of SbAPX was similar to that of other APXs, SbAPX uniquely displayed four bound ascorbates rather than one. In addition to the ɣ-heme pocket identified in other APXs, ascorbates were bound at the δ-meso and two solvent-exposed pockets. Consistent with the presence of multiple binding sites, our results indicated that the H2O2-dependent oxidation of ascorbate displayed positive cooperativity. Bound ascorbate at two surface sites established an intricate proton network with ascorbate at the ɣ-heme edge and δ-meso sites. Based on crystal structures, steady-state kinetics, and site-directed mutagenesis results, both ascorbate molecules at the ɣ-heme edge and the one at the surface are expected to participate in the oxidation reaction. We provide evidence that the H2O2-dependent oxidation of ascorbate by APX produces a C2-hydrated bicyclic hemiketal form of dehydroascorbic acid at the ɣ-heme edge, indicating two successive electron transfers from a single-bound ascorbate. In addition, the δ-meso site was shared with several organic compounds, includingp-coumaric acid and other phenylpropanoids, for the potential radicalization reaction. Site-directed mutagenesis of the critical residue at the ɣ-heme edge (R172A) only partially reduced polymerization activity. Thus, APX removes stress-generated H2O2with ascorbates, and also uses this same H2O2to potentially fortify cell walls via oxidative polymerization of phenylpropanoids in response to stress.