A Minimized Chemoenzymatic Cascade for Bacterial Luciferase in Bioreporter Applications

Abstract

Bacterial luciferase (Lux) catalyzes a bioluminescence reaction by using long-chain aldehyde, reduced flavin and molecular oxygen as substrates. The reaction can be applied in reporter gene systems for biomolecular detection in both prokaryotic and eukaryotic organisms. Because reduced flavin is unstable under aerobic conditions, another enzyme, flavin reductase, is needed to supply reduced flavin to the Lux-catalyzed reaction. To create a minimized cascade for Lux that would have greater ease of use, a chemoenzymatic reaction with a biomimetic nicotinamide (BNAH) was used in place of the flavin reductase reaction in the Lux system. The results showed that the minimized cascade reaction can be applied to monitor bioluminescence of the Lux reporter in eukaryotic cells effectively, and that it can achieve higher efficiencies than the system with flavin reductase. This development is useful for future applications as high-throughput detection tools for drug screening applications.

Divorce in the two-component BVMO family: the single oxygenase for enantioselective chemo-enzymatic Baeyer–Villiger oxidations

“…20 NCB-driven approaches were documented for other flavoenzymes, such as very recently for a regioselective aromatic hydroxylation, 41 and other two-component FMPOs. [20][21][22] These systems outperformed the natural enzymatic regeneration system proceeding at four times faster initial reaction rates 21 and NCBs showed a very high K m compared with the natural nicotinamide cofactor. 41 However, they have never applied to type II BVMO.…”

“…16 Moreover, they have been established to replace their natural templates for numerous flavin-dependent enzymes [17][18][19] with a flavin shuttled electron mediation. [20][21][22] Most of the studies on enzymatic BV oxidation focused on one-component FPMOs from group B (also known as type I BVMOs). These enzymes are available in high numbers in protein databanks, easily detectable by consensus sequence research 23 and subsequently more frequently investigated and applied in many catalytic processes.…”

Flavoprotein Monooxygenases and Halogenases

“…The crystal structure of luciferase from Vibrio harveyi [109] paved the way for structurefunction relationship studies of FPMOs with a luciferase-like (β/α)8 domain (Figure 7.1; CATH code 3.20.20.30). The bioluminescence reaction of bacterial luciferase can be applied in prokaryotic and eukaryotic reporter gene systems for the detection of a wide range of specific analytes [110,111]. LadA monooxygenases from thermophilic Geobacillus species (EC 1.14.14.28) catalyze the hydroxylation of long-chain alkanes to the corresponding primary alcohols [112].…”

“…In recent years, synthetic nicotinamide coenzyme biomimetics (NCBs), appealing for their cost efficiency [189][190][191], have been coupled to various FPMOs to replace NAD(P)H as electron donor. NCBs have been found active with group A hydroxylases [192], group C luciferases [110], group E SMOs [136],…”

Asymmetric azidohydroxylation of styrene derivatives mediated by a biomimetic styrene monooxygenase enzymatic cascade

“…Non-enzymatic approaches have been investigated by shortcutting NADH/StyB using various photo-or electrochemical methods to reduce FAD. [24][25][26][27][28][29] In particular, 1benzyl-1,4-dihydronicotinamide (BNAH) was shown to be effective and simple to use as a reductant with StyA 16,30 and other two-component flavoprotein monooxygenases such as halogenases, 31 a bacterial luciferase, 32 and an FMN-dependent type II Baeyer-Villiger monooxygenase. 33 BNAH thus circumvents the use of two enzymes (StyB, dehydrogenase) and NADH, for using oxygenase StyA in biocatalytic reactions (Figure 1B).…”