Oba Yuichi, Tanaka Kiichi, Inouye Satoshi
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, Japan.
Biosci Biotechnol Biochem. 2006 Nov;70(11):2739-44. doi: 10.1271/bbb.60364. Epub 2006 Nov 7.
Firefly luciferase and fatty acyl-CoA synthetase are members of the acyl-CoA synthetase super family, which consists of a large N-terminal domain and a small C-terminal domain. Previously we found that firefly luciferase has fatty acyl-CoA synthetic activity, and also identified that the homolog of firefly luciferase in Drosophila melanogaster (CG6178) is a fatty acyl-CoA synthetase and is not a luciferase. In this study, we constructed chimeric proteins by exchanging the domain between Photinus pyralis luciferase (PpLase) and Drosophila CG6178, and determined luminescence and fatty acyl-CoA synthetic activities. A chimeric protein with the N-terminal domain of PpLase and the C-terminal domain of CG6178 (Pp/Dm) had luminescence activity, showing approximately 4% of the activity of wild-type luciferase. The Pp/Dm protein also had fatty acyl-CoA synthetic activity and the substrate specificity was similar to PpLase. In contrast, a chimeric protein with the N-terminal domain of CG6178 and the C-terminal of PpLase (Dm/Pp) had only fatty acyl-CoA synthetase activity, and the substrate specificity was similar to CG6178. These results suggest that the N-terminal domain of firefly luciferase is essential for substrate recognition, and that the C-terminal domain is indispensable but not specialized for the luminescence reaction.
萤火虫荧光素酶和脂肪酰辅酶A合成酶是酰基辅酶A合成酶超家族的成员，该家族由一个大的N端结构域和一个小的C端结构域组成。此前我们发现萤火虫荧光素酶具有脂肪酰辅酶A合成活性，并且还鉴定出果蝇（CG6178）中萤火虫荧光素酶的同源物是一种脂肪酰辅酶A合成酶而非荧光素酶。在本研究中，我们通过交换萤火虫荧光素酶（PpLase）和果蝇CG6178之间的结构域构建了嵌合蛋白，并测定了发光和脂肪酰辅酶A合成活性。一种具有PpLase的N端结构域和CG6178的C端结构域的嵌合蛋白（Pp/Dm）具有发光活性，其活性约为野生型荧光素酶的4%。Pp/Dm蛋白也具有脂肪酰辅酶A合成活性，且底物特异性与PpLase相似。相反，一种具有CG6178的N端结构域和PpLase的C端的嵌合蛋白（Dm/Pp）仅具有脂肪酰辅酶A合成酶活性，且底物特异性与CG6178相似。这些结果表明，萤火虫荧光素酶的N端结构域对于底物识别至关重要，而C端结构域是不可或缺的，但并非专门用于发光反应。
