Viviani V R, Arnoldi F G C, Venkatesh B, Neto A J S, Ogawa F G T, Oehlmeyer A T L, Ohmiya Y
Universidade Federal de Sao Carlos, Campus de Sorocaba, Sorocaba, SP, Brazil.
J Biochem. 2006 Oct;140(4):467-74. doi: 10.1093/jb/mvj190. Epub 2006 Sep 8.
The luciferases of the railroad worm Phrixotrix (Coleoptera: Phengodidae) are the only beetle luciferases that naturally produce true red bioluminescence. Previously, we cloned the green- (PxGR) and red-emitting (PxRE) luciferases of railroad worms Phrixotrix viviani and P. hirtus[OLE1]. These luciferases were expressed and purified, and their active-site properties were determined. The red-emitting PxRE luciferase displays flash-like kinetics, whereas PxGR luciferase displays slow-type kinetics. The substrate affinities and catalytic efficiency of PxRE luciferase are also higher than those of PxGR luciferase. Fluorescence studies with 8-anilino-1-naphthalene sulfonic acid and 6-p-toluidino-2-naphthalene sulfonic acid showed that the PxRE luciferase luciferin-binding site is more polar than that of PxGR luciferase, and it is sensitive to guanidine. Mutagenesis and modelling studies suggest that several invariant residues in the putative luciferin-binding site of PxRE luciferase cannot interact with excited oxyluciferin. These results suggest that one portion of the luciferin-binding site of the red-emitting luciferase is tighter than that of PxGR luciferase, whereas the other portion could be more open and polar.
铁路蠕虫（鞘翅目：萤火虫科）的荧光素酶是唯一能自然产生真红色生物发光的甲虫荧光素酶。此前，我们克隆了维氏铁路蠕虫（Phrixotrix viviani）和多毛铁路蠕虫（P. hirtus）的绿色荧光素酶（PxGR）和红色荧光素酶（PxRE）[OLE1]。这些荧光素酶得到了表达和纯化，并测定了它们的活性位点特性。发出红色光的PxRE荧光素酶表现出闪光样动力学，而PxGR荧光素酶表现出慢型动力学。PxRE荧光素酶的底物亲和力和催化效率也高于PxGR荧光素酶。用8-苯胺基-1-萘磺酸和6-对甲苯胺基-2-萘磺酸进行的荧光研究表明，PxRE荧光素酶的荧光素结合位点比PxGR荧光素酶的更具极性，并且对胍敏感。诱变和建模研究表明，PxRE荧光素酶假定的荧光素结合位点中的几个不变残基不能与激发态氧化荧光素相互作用。这些结果表明，发出红色光的荧光素酶的荧光素结合位点的一部分比PxGR荧光素酶的更紧密，而另一部分可能更开放且更具极性。
