Masuda Shinji, Tomida Yoshiyuki, Ohta Hiroyuki, Takamiya Ken-Ichiro
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.
J Mol Biol. 2007 May 18;368(5):1223-30. doi: 10.1016/j.jmb.2007.02.087. Epub 2007 Mar 6.
AppA is a novel blue-light receptor that controls photosynthetic gene expression in the purple bacterium Rhodobacter sphaeroides. The photocycle reaction of the light-sensing domain, BLUF, is unique in the sense that a few hydrogen bond rearrangements are accompanied by only slight structural changes of the bound chromophore. However, the exact features of the hydrogen bond network around the active site are still the subject of some controversy. Here we present biochemical and genetic evidence showing that either Gln63 or Trp104 in the active site of the BLUF domain is crucial for light sensing, which in turn controls the antirepressor activity of AppA. Specifically, the Q63L and W104A mutants of AppA are insensitive to blue light in vivo and in vitro, and their activity is similar to that of the light-adapted wild-type AppA. Based on spectroscopic and structural information described previously, we conclude that light-dependent formation and breakage of the hydrogen bond between Gln63 and Trp104 are critical for the light-sensing mechanism of AppA.
AppA是一种新型蓝光受体，可控制紫色细菌球形红杆菌中的光合基因表达。光感应结构域BLUF的光循环反应具有独特性，即少数氢键重排仅伴随着结合发色团的轻微结构变化。然而，活性位点周围氢键网络的确切特征仍然存在一些争议。在此，我们提供生化和遗传学证据表明，BLUF结构域活性位点中的Gln63或Trp104对于光感应至关重要，进而控制AppA的抗阻遏物活性。具体而言，AppA的Q63L和W104A突变体在体内和体外对蓝光均不敏感，其活性与光适应的野生型AppA相似。基于先前描述的光谱和结构信息，我们得出结论，Gln63和Trp104之间氢键的光依赖性形成和断裂对于AppA的光感应机制至关重要。
