Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance
Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance
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DOI：
10.1042/bj20051847
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发表时间：
2006-07-15
影响因子：
4.1
通讯作者：
Murray, James A. H.
中科院分区：
文献类型：
--
作者：
Law, G. H. Erica;Gandelman, Olga A.;Murray, James A. H.
萤火虫荧光素酶以 ATP-Mg2+、萤火虫荧光素和分子氧为底物催化两步反应，从而有效发射黄绿光。我们报告了新型荧光素酶突变体的鉴定，该突变体结合了改进的 pH 耐受性和热稳定性，并保留了野生型酶的比活性。这些是通过将暴露于溶剂的萤火虫荧光素酶中的非保守疏水性氨基酸诱变为亲水性残基，然后进行体内活性筛选来鉴定的。发现突变体 F14R、L35Q、V182K、1232K 和 F465R 是各自位置的优选取代。这些氨基酸取代的效果是相加的，因为五种取代的组合产生了一种具有极大改善的 pH 耐受性和高达 45°C 稳定性的酶。所有突变体，包括具有所有五种取代的突变体，相对于重组野生型酶，既没有表现出比活性的降低，也没有表现出动力学常数的任何实质性差异。预计是合并的。在许多体外和体内应用中，突变体将优于野生型荧光素酶。
Firefly luciferase catalyses a two-step reaction, using ATP-Mg2+, firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow-green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specific activity of the wild-type enzyme. These were identified by the mutagenesis of solvent-exposed non-conserved hydrophobic, amino acids to hydrophilic residues in Photinus pyralis firefly luciferase followed by in vivo activity screening. Mutants F14R, L35Q, V182K, 1232K and F465R were found to be the preferred substitutions at the respective positions. The effects of these amino acid replacements are additive, since combination of the five substitutions produced an enzyme with greatly improved pH-tolerance and stability up to 45 degrees C. All mutants, including the mutant with all five substitutions, showed neither a decrease in specific activity relative to the recombinant wild-type enzyme, nor any substantial differences in kinetic constants. It is envisaged that the combined. mutant will be superior to wild-type luciferase for many in vitro and in vivo applications.