Sedláček Vojtěch, Kryl Martin, Kučera Igor
Department of Biochemistry, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic.
Antioxidants (Basel). 2022 May 3;11(5):902. doi: 10.3390/antiox11050902.
ArsH is encoded by two identical genes located in two distinct putative arsenic resistance () operons. -produced recombinant N-His-ArsH was characterized both structurally and kinetically. The X-ray structure of ArsH revealed a flavodoxin-like domain and motifs for the binding of flavin mononucleotide (FMN) and reduced nicotinamide adenine dinucleotide phosphate (NADPH). The protein catalyzed FMN reduction by NADPH via ternary complex mechanism. At a fixed saturating FMN concentration, it acted as an NADPH-dependent organoarsenic reductase displaying ping-pong kinetics. A 1:1 enzymatic reaction of phenylarsonic acid with the reduced form of FMN (FMNH) and formation of phenylarsonous acid were observed. Growth experiments with and revealed increased toxicity of phenylarsonic acid to cells expressing , which may be related to in vivo conversion of pentavalent As to more toxic trivalent form. ArsH expression was upregulated not only by arsenite, but also by redox-active agents paraquat, tert-butyl hydroperoxide and diamide. A crucial role is played by the homodimeric transcriptional repressor ArsR, which was shown in in vitro experiments to monomerize and release from the DNA-target site. Collectively, our results establish ArsH as responsible for enhancement of organo-As(V) toxicity and demonstrate redox control of operon.
ArsH由位于两个不同的假定抗砷（）操纵子中的两个相同基因编码。对产生的重组N - His - ArsH进行了结构和动力学表征。ArsH的X射线结构显示出一个类黄素氧还蛋白结构域以及结合黄素单核苷酸（FMN）和还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的基序。该蛋白通过三元复合物机制催化NADPH还原FMN。在固定的饱和FMN浓度下，它作为一种依赖NADPH的有机砷还原酶，呈现乒乓动力学。观察到苯胂酸与还原型FMN（FMNH）的1:1酶促反应以及苯亚胂酸的形成。用和进行的生长实验表明，苯胂酸对表达的细胞毒性增加，这可能与五价砷在体内转化为毒性更强的三价形式有关。ArsH的表达不仅受亚砷酸盐上调，还受氧化还原活性剂百草枯、叔丁基过氧化氢和二酰胺上调。同二聚体转录阻遏物ArsR起着关键作用，体外实验表明它会单体化并从DNA靶位点释放。总的来说，我们的结果确定ArsH是导致有机砷（V）毒性增强的原因，并证明了操纵子的氧化还原调控。
