C-terminal residues of ferredoxin-NAD(P)+ reductase from Chlorobaculum tepidum are responsible for reaction dynamics in the hydride transfer and redox equilibria with NADP+/NADPH
C-terminal residues of ferredoxin-NAD(P)+ reductase from Chlorobaculum tepidum are responsible for reaction dynamics in the hydride transfer and redox equilibria with NADP+/NADPH
复制标题
来自温热绿杆菌的铁氧还蛋白-NAD(P) 还原酶的 C 端残基负责氢化物转移和与 NADP /NADPH 的氧化还原平衡中的反应动力学
DOI：
10.1007/s11120-017-0462-z
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发表时间：
2017
影响因子：
3.7
通讯作者：
Asano Tomoya
中科院分区：
文献类型：
--
作者：
Seo Daisuke;Asano Tomoya
天青绿杆菌铁氧还蛋白-NAD(P)+还原酶([EC 1.18.1.2]，[EC 1.18.1.3])与细菌NADPH-硫氧还蛋白还原酶(TrxR)在结构上是同源的，但相对于TrxR有一个独特的C-末端延伸，它与黄素腺嘌呤二核苷酸修复基团的异烟肼环部分相互作用。在这项研究中，我们引入了C-末端残基的截断，通过光谱分析和动力学分析考察了它们在CtFNR与NADP+和NADPH反应中的作用。从Tyr326到Glu360(整个C-末端延伸区)、从Phe337到Glu360(省略了异烟肼环表面的Phe337)和从Ser338到Glu360(保持Phe337完整)的残基截断导致黄素吸收带蓝移。在稳态分析中，截断导致对NADP+的解离常数略有增加，对NADPH的米氏常数略有降低。NADPH氧化还原反应的稳态前研究表明，与野生型CtFNR相比，缺失Tyr326-Glu360降低了氢化物转移速率，平衡时还原酶的量增加。相反，Phe337-Glu360和Ser338-Glu360的缺失仅导致反应动力学和氧化还原平衡的微小变化。这些结果表明，CtFNR的C-末端区域负责电荷转移络合物的形成和稳定性，导致氧化还原性质和对NADP+/NADPH的反应活性的改变。
Ferredoxin-NAD(P)+reductase ([EC 1.18.1.2], [EC 1.18.1.3]) fromChlorobaculum tepidum(CtFNR) is structurally homologous to the bacterial NADPH-thioredoxin reductase (TrxR), but possesses a unique C-terminal extension relative to TrxR that interacts with the isoalloxazine ring moiety of the flavin adenine dinucleotide prosthetic group. In this study, we introduce truncations to the C-terminal residues to examine their role in the reactions ofCtFNR with NADP+and NADPH by spectroscopic and kinetic analyses. The truncation of the residues from Tyr326 to Glu360 (the whole C-terminal extension region), from Phe337 to Glu360 (omitting Phe337 on there-face of the isoalloxazine ring) and from Ser338 to Glu360 (leaving Phe337 intact) resulted in a blue-shift of the flavin absorption bands. The truncations caused a slight increase in the dissociation constant toward NADP+and a slight decrease in the Michaelis constant toward NADPH in steady-state assays. Pre-steady-state studies of the redox reaction with NADPH demonstrated that deletions of Tyr326–Glu360 decreased the hydride transfer rate, and the amount of reduced enzyme increased at equilibrium relative to wild-typeCtFNR. In contrast, the deletions of Phe337–Glu360 and Ser338–Glu360 resulted in only slight changes in the reaction kinetics and redox equilibrium. These results suggest that the C-terminal region ofCtFNR is responsible for the formation and stability of charge-transfer complexes, leading to changes in redox properties and reactivity toward NADP+/NADPH.