Pyrearinus termitilluminans larval click beetle luciferase: active site properties, structure and function relationships and comparison with other beetle luciferases
Pyrearinus termitilluminans larval click beetle luciferase: active site properties, structure and function relationships and comparison with other beetle luciferases
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DOI：
10.1039/b9pp00053d
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发表时间：
2009-01-01
影响因子：
3.1
通讯作者：
Viviani, V. R.
中科院分区：
文献类型：
--
作者：
Neto, A. J. Silva;Scorsato, V.;Viviani, V. R.
几种甲虫荧光素酶已被克隆并测序。然而，大多数关于结构和功能关系以及生物分析应用的研究都是使用对 pH 敏感的萤火虫荧光素酶进行的。几年前，我们克隆了 Pyrearinus termitluminans 幼虫点击甲虫荧光素酶，它在甲虫荧光素酶中显示出最蓝移的生物发光，并且对 pH 值不敏感。该酶在大肠杆菌中表达、纯化，并研究了其特性。该荧光素酶显示出较慢的发光动力学、与其他甲虫荧光素酶相当的 K-M 值和较高的催化常数。 8-苯胺基-1-萘磺酸 (1,8-ANS) 的荧光研究和建模研究表明，该荧光素酶的荧光素结合位点非常疏水，支持溶剂和取向极化效应作为生物发光颜色的决定机制。尽管在 pH 6-8 范围内 pH 不敏感，但在 pH 10 时，该荧光素酶表现出显着的红移和生物发光光谱的展宽。模型研究表明残基 C312 可能在生物发光颜色调节中发挥重要作用。与其他甲虫荧光素酶相比，Pyrearinus termitluminans 荧光素酶在细菌细胞环境中也表现出更高的热稳定性和持续发光，这使得该荧光素酶特别适合体内细胞分析和生物成像。
Several beetle luciferases have been cloned and sequenced. However, most studies on structure and function relationships and bioanalytical applications were done with firefly luciferases, which are pH sensitive. Several years ago we cloned Pyrearinus termitilluminans larval click beetle luciferase, which displays the most blue-shifted bioluminescence among beetle luciferases and is pH insensitive. This enzyme was expressed in E. coli, purified, and its properties investigated. This luciferase shows slower luminescence kinetics, K-M values comparable to other beetle luciferases and high catalytic constant. Fluorescence studies with 8-anilino-1-naphtalene-sulfonic acid (1,8-ANS) and modeling studies suggest that the luciferin binding site of this luciferase is very hydrophobic, supporting the solvent and orientation polarizability effects as determining mechanisms for bioluminescence colors. Although pH insensitive in the range between pH 6-8, at pH 10 this luciferase displays a remarkable red-shift and broadening of the bioluminescence spectrum. Modeling studies suggest that the residue C312 may play an important role in bioluminescence color modulation. Compared to other beetle luciferases, Pyrearinus termitilluminans luciferase also displays higher thermostability and sustained luminescence in a bacterial cell environment, which makes this luciferase particularly suitable for in vivo cell analysis and bioimaging.