Biosynthesis-Inspired Deracemizative Production of D-Luciferin In Vitro by Combining Luciferase and Thioesterase

Abstract

Due to the strict enantioselectivity of firefly luciferase (FLuc), only D-luciferin can be used as a substrate for the bioluminescence (BL) reaction. Unfortunately, luciferin racemizes easily and accumulation of nonluminous L-luciferin has negative influences on the light-emitting reaction. By a detailed analysis of luciferin chirality, however, it becomes clarified that L-luciferin is the biosynthetic precursor of D-luciferin in fireflies and undergoes the enzymatic chiral inversion. By the chiral inversion reaction, the enantiopurity of luciferin can be maintained in the reaction mixture for applications using FLuc. Thus, chirality is crucial for the BL reaction and essential for investigating and applying the biosynthesis of D-luciferin. Here, we describe the methods for the analysis of chiral inversion reaction using high-performance liquid chromatography (HPLC) with a chiral column. We also introduce an example of an in vitro deracemizative BL reaction system using a combination of FLuc and fatty acyl-CoA thioesterase, which is inspired by the chiral inversion mechanism in the biosynthetic pathway of D-luciferin.