Single Amino Acid Mutation Decouples Photochemistry of the BLUF Domain from the Enzymatic Function of OaPAC and Drives the Enzyme to a Switched-on State
Single Amino Acid Mutation Decouples Photochemistry of the BLUF Domain from the Enzymatic Function of OaPAC and Drives the Enzyme to a Switched-on State
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DOI：
10.1016/j.jmb.2023.168312
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发表时间：
2024-03-03
影响因子：
5.6
通讯作者：
Lukacs，Andras
中科院分区：
文献类型：
--
作者：
Collado，Jinnette Tolentino;Bodis，Emoke;Lukacs，Andras
光活化腺苷酸环化酶（PAC）是结合BLUF（使用黄素的蓝光）结构域和腺苷酸环化酶结构域的联合收割机，其能够在蓝光激发时增加重要的第二信使cAMP（环磷酸腺苷）的水平。光诱导的BLUF结构域的变化通过一种尚未建立的机制转导到腺苷酸环化酶结构域。在BLUF结构域的光活化机制中，存在于黄素附近的一个关键残基是靠近黄素的N5的谷氨酰胺氨基酸。这种残留物的作用已被广泛的实验和理论研究。然而，其在光活化腺苷酸环化酶（OaPAC）的活性中的作用从未得到解决。在这项工作中，我们应用超快瞬态可见和红外光谱研究Q48E OaPAC突变体的光化学。这种突变改变了初级电子转移过程，并将酶转换为永久的“开启”状态，与野生型OaPAC暗适应状态的cAMP水平相比，能够在黑暗条件下增加cAMP水平。差示扫描量热法测量指向Q48E OaPAC突变体的不太紧凑的结构。这些发现的集合提供了对PAC中重要元素的深入了解，以及它们的微调如何有助于光遗传学设备的设计。
Photoactivated adenylate cyclases (PACs) are light-activated enzymes that combine a BLUF (blue-light using flavin) domain and an adenylate cyclase domain that are able to increase the levels of the important second messenger cAMP (cyclic adenosine monophosphate) upon blue-light excitation. The light-induced changes in the BLUF domain are transduced to the adenylate cyclase domain via a mechanism that has not yet been established. One critical residue in the photoactivation mechanism of BLUF domains, present in the vicinity of the flavin is the glutamine amino acid close to the N5 of the flavin. The role of this residue has been investigated extensively both experimentally and theoretically. However, its role in the activity of the photoactivated adenylate cyclase, OaPAC has never been addressed. In this work, we applied ultrafast transient visible and infrared spectroscopies to study the photochemistry of the Q48E OaPAC mutant. This mutation altered the primary electron transfer process and switched the enzyme into a permanent ‘on’ state, able to increase the cAMP levels under dark conditions compared to the cAMP levels of the dark-adapted state of the wild-type OaPAC. Differential scanning calorimetry measurements point to a less compact structure for the Q48E OaPAC mutant. The ensemble of these findings provide insight into the important elements in PACs and how their fine tuning may help in the design of optogenetic devices.