Department of Chemistry and Bioscience, Graduate School of Science and Engineering, Kagoshima University, Japan.
Department of Chemistry and Bioscience, Graduate School of Science and Engineering, Kagoshima University, Japan.
Biochim Biophys Acta Gen Subj. 2017 Aug;1861(8):2112-2118. doi: 10.1016/j.bbagen.2017.04.010. Epub 2017 Apr 25.
Due to the strict enantioselectivity of firefly luciferase, only d-luciferin can be used as a substrate for bioluminescence reactions. Unfortunately, luciferin racemizes easily and accumulation of nonluminous l-luciferin has negative influences on the light emitting reaction. Thus, maintaining the enantiopurity of luciferin in the reaction mixture is one of the most important demands in bioluminescence applications using firefly luciferase. In fireflies, however, l-luciferin is the biosynthetic precursor of d-luciferin, which is produced from the L-form undergoing deracemization. This deracemization consists of three successive reactions: l-enantioselective thioesterification by luciferase, in situ epimerization, and hydrolysis by thioesterase. In this work, we introduce a deracemizative luminescence system inspired by the biosynthetic pathway of d-luciferin using a combination of firefly luciferase from Luciola cruciata (LUC-G) and fatty acyl-CoA thioesterase II from Escherichia coli (TESB). The enzymatic reaction property analysis indicated the importance of the concentration balance between LUC-G and TESB for efficient d-luciferin production and light emission. Using this deracemizative luminescence system, a highly sensitive quantitative analysis method for l-cysteine was constructed. This LUC-G-TESB combination system can improve bioanalysis applications using the firefly bioluminescence reaction by efficient deracemization of D-luciferin.
由于萤火虫荧光素酶具有严格的对映选择性，只有 d-荧光素才能作为生物发光反应的底物。然而，不幸的是，荧光素容易外消旋化，并且非发光的 l-荧光素的积累对发光反应有负面影响。因此，在使用萤火虫荧光素酶的生物发光应用中，保持反应混合物中荧光素的对映体纯度是最重要的要求之一。然而，在萤火虫中，l-荧光素是 d-荧光素的生物合成前体，d-荧光素是由经历外消旋化的 L 型产生的。这种外消旋化由三个连续的反应组成：荧光素酶的 l-对映选择性硫酯化、原位差向异构化和硫酯酶的水解。在这项工作中，我们受 d-荧光素生物合成途径的启发，引入了一种由来自 Luciola cruciata 的荧光素酶（LUC-G）和大肠杆菌的脂肪酸酰基辅酶 A 硫酯酶 II（TESB）组合而成的外消旋发光系统。酶促反应性质分析表明，LUC-G 和 TESB 之间浓度平衡对于高效生产 d-荧光素和发光的重要性。使用这种外消旋发光系统，构建了一种用于 l-半胱氨酸的高灵敏度定量分析方法。这种 LUC-G-TESB 组合系统可以通过 d-荧光素的高效外消旋化来改善使用萤火虫生物发光反应的生物分析应用。
