Single Amino Acid Substitution Reveals Latent Photolyase Activity in Arabidopsis cry1
Single Amino Acid Substitution Reveals Latent Photolyase Activity in Arabidopsis cry1
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DOI：
10.1002/anie.201203476
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发表时间：
2012-01-01
影响因子：
16.6
通讯作者：
Ahmad, Margaret
中科院分区：
文献类型：
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作者：
Burney, Sarah;Wenzel, Ringo;Ahmad, Margaret
隐色素是在整个生物王国中发现的黄蛋白受体。在脊椎动物中，隐花色素在生物钟中起作用，与人类癌症有关，并被认为是候鸟中的磁感受器。所有隐花色素的特征在于它们与光激活的DNA修复酶，光解酶的惊人结构相似，尽管它们广泛存在和不同的信号作用。[1-5]与光解酶一样，隐花色素在疏水口袋中结合吸收光的黄素辅因子（FAD），并响应于光进行蛋白质内电子转移和光还原。[6，7]然而，与光解酶不同，隐花色素在植物和动物中具有已知的信号作用，并且不修复DNA。信号所需的区别特征的本质仍然难以捉摸。最近的研究表明，动物和植物隐花色素在黑暗中积累氧化（OX）黄素，并在光照下形成半还原自由基形式（SR），而在相同条件下的光解酶在黑暗中积累完全还原的阴离子黄素（RED），这是DNA修复所需的。[8-10]还表明植物和昆虫隐花色素的SR形式与生物活性相关。[8，9，11]虽然黄素氧化态的功能意义仍在讨论中，[12]具有已知信号作用的隐花色素和修复DNA的光解酶之间的关键区别是体内结合黄素的氧化态。在这里，我们探讨了黄素氧化还原状态可能提供一个线索，植物和动物隐花色素是如何从祖先的光解酶。从机理上讲，黄素的质子化可能是由拟南芥cry 1（Atcry 1）396位保守氨基酸引起的，Atcry 1在所有植物隐花色素中都是带负电荷的天冬氨酸（D）残基，而在E.大肠杆菌和其它光解酶中，这是一个中性天冬酰胺（N）或带正电荷的赖氨酸（K）残基。[13]在隐花色素中，该位置的D残基被认为是光照时黄素的可能质子供体[14，15]，并且可以解释氧化还原电位的差异，因此，有利于黄素氧化还原状态。为了证明氧化还原状态可能确实是隐花色素和光解酶之间的决定性区别，将突变D396 N引入Atcry 1中，并从杆状病毒表达系统分离纯化的重组蛋白（图1）。纯化的D396 N突变蛋白与野生型蛋白一样结合OX黄素（图1a，图1，2;在光照前t0=黑暗）。吸收光谱在D突变为N时保持不变（图1a，图1、2），与D396在黑暗中质子化一致。[15]在有氧条件下照射后，在温和还原剂（10 mm β-巯基乙醇（BME））的存在下，通过500- 600 nm之间增加的吸光度检测到向SR（FADH 8）形式黄素的转变。随着进一步的照射，可以看到完全还原的FADHlO（RED）氧化还原形式的显著形成，因为在450 nm处的吸光度持续降低，而在500- 600 nm处没有增加（图1a，图1，3）。在这些相同的光照条件下，野生型蛋白质仅略微减少，不能积累RED形式，并且也积累少得多的SR形式（图1a，图2，4）。因此，突变蛋白D396 N的光照导致形成RED黄素，其对DNA修复有用，而不是SR黄素，其与隐花色素相关
Cryptochromes are flavoprotein receptors found throughout the biological kingdom. In vertebrates, cryptochromes function in the circadian clock, are linked to human cancers, and have been proposed as magnetoreceptors in migratory birds. All cryptochromes are characterized by their striking structural similarity to light-activated DNA-repair enzymes, photolyases, despite their widespread occurrence and different signaling roles.[1–5] Like photolyases, cryptochromes bind a light-absorbing flavin cofactor (FAD) in a hydrophobic pocket and undergo intraprotein electron transfer and photoreduction in response to light.[6, 7] However, unlike photolyases, cryptochromes have known signaling roles in plants and animals and do not repair DNA. The nature of the distinguishing characteristics required for signaling has remained elusive. It has recently been shown that animal and plant cryptochromes accumulate oxidized (OX) flavin in the dark and form the semi-reduced radical form (SR) upon illumination, whereas photolyases under the same conditions accumulate fully reduced anionic flavin (RED) in the dark, which is required for DNA repair.[8–10] It has also been shown that the SR form of plant and insect cryptochromes is correlated with biological activity.[8, 9, 11] Although the functional significance of the flavin oxidation state is still under discussion,[12] a critical difference between cryptochromes with known signaling roles and photolyases that repair DNA is the oxidation state of bound flavin in vivo. Herein, we explore how the flavin redox state may provide a clue as to how plant and animal cryptochromes evolved from ancestral photolyases. Mechanistically, protonation of flavin may result from a conserved amino acid at position 396 of Arabidopsis cry1 (Atcry1), which is a negatively charged aspartic acid (D) residue in all plant cryptochromes, whereas in E. coli and other photolyases this is a neutral asparagine (N) or positively charged lysine (K) residue.[13] In cryptochromes, the D residue at this position has been suggested as a possible proton donor for flavin upon illumination [14, 15] and may explain the difference in redox potentials and hence, the favored flavin redox states. To demonstrate that redox state may indeed be a defining distinction between cryptochromes and photolyases, the mutation D396N was introduced into Atcry1 and the purified recombinant protein isolated from a baculovirus expression system (Figure 1).The purified D396N mutant protein binds OX flavin as is the case for wild-type protein (Figure 1a, panels 1, 2; before illumination t0= dark). The absorption spectra remain unchanged by the mutation of D to N (Figure 1a, panels 1, 2), consistent with D396 being protonated in the dark.[15] Upon illumination under aerobic conditions, in the presence of a mild reducing agent (10 mm β-mercaptoethanol (BME)), transition to the SR (FADH8) form of flavin was detected by increased absorbance between 500–600nm. With further illumination, significant formation of the fully reduced FADHÀ (RED) redox form can be seen because of a continuing decrease in absorbance at 450 nm, without an increase at 500–600nm (Figure1a, panels 1, 3). Under these same illumination conditions, wild-type protein was only slightly reduced, failed to accumulate the RED form, and also accumulated far less of the SR form (Figure 1a, panels 2, 4). Thus, illumination of the mutant protein D396N results in formation of the RED flavin, useful for DNA repair, rather than the SR flavin, which is correlated with cryptochrome