Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer

Using the lux operon (luxCDABE) of bacterial bioluminescence system as an autonomous luminous reporter has been demonstrated in bacteria, plant and mammalian cells. However, applications of bacterial bioluminescence-based imaging have been limited because of its low brightness. Here, we engineered the bacterial luciferase (heterodimer of luxA and luxB) by fusion with Venus, a bright variant of yellow fluorescent protein, to induce bioluminescence resonance energy transfer (BRET). By using decanal as an externally added substrate, color change and ten-times enhancement of brightness was achieved in Escherichia coli when circularly permuted Venus was fused to the C-terminus of luxB. Expression of the Venus-fused luciferase in human embryonic kidney cell lines (HEK293T) or in Nicotiana benthamiana leaves together with the substrate biosynthesis-related genes (luxC, luxD and luxE) enhanced the autonomous bioluminescence. We believe the improved luciferase will forge the way towards the potential development of autobioluminescent reporter system allowing spatiotemporal imaging in live cells.

ВЛИЯНИЕ ОСМОЛИТОВ НА БИОЛЮМИНЕСЦЕНТНУЮ РЕАКЦИЮ БАКТЕРИЙ: СТРУКТУРНО-ДИНАМИЧЕСКИЕ АСПЕКТЫ

The effects of viscous media with glycerol and sucrose (10-40%) on the kinetics of the bacterial bioluminescent reaction have been investigated by stopped-flow technique. Increment of quantum yield in media with 10% of both osmolytes was shown. Higher concentrations of glycerol, up to 30-40%, were found to reduce the efficiency of the reaction, while this effect was not observed in the media with sucrose. The molecular dynamics simulation was used to study the structure of bacterial luciferase surrounded by either water molecules solely or by mixture of water with various numbers of glycerol/sucrose molecules. It was found that both cosolvents at studied concentrations did not cause a significant change in conformation of bacterial luciferase. The calculated root mean square fluctuations for Cα-atoms of bacterial luciferase α-subunit indicated that the higher flexibility of the enzyme mobile loop could be responsible for increment of quantum yield in the presence of 10% of both osmolytes. The active site of bacterial luciferase was found to be accessible for glycerol molecules while sucrose did not enter catalytic gorge. Moreover, at 30% and 40% concentration the glycerol molecules were found to locate in the active site of bacterial luciferase throughout the whole simulation time (40 ns) and to exclude water molecules, which can explain the experimentally obtained reaction quantum yield decrease.

Structure, Enzymatic Mechanism of Action, Applications, Advantages, Disadvantages and Modifications of Luciferase Enzyme

North American Firefly, Photinu spyralis, entertainment countless observers, probably because of human arrival mainland. Between the period of 1950s and 1980s, many young biologists spent their money as a firefly collector, first hired by a Professor William D McElroy at Johns Hopkins University, later became a member of the Sigma Firefly Club. In 1985, when Marlene DeLuca and her colleagues cloned cDNA encoding luciferase, a replacement source of the enzyme becomes available and later in labs around the world, many other organisms began to emit unique yellow-green glow as a result of firefly expression Luciferase is in their cells. Now, luciferase and its genes have become very useful for research purposes and also for a variety of commercial purposes. The structure of firefly and bacterial luciferase will be of great value for the development of applications in many processes. There are many firefly luciferase homologues that can catalyze similar reactions with similar amino acid sequences. Several applications are described in different publications on bioluminescence and chemiluminescence. Firefly flicker is always very interesting to observe it. Firefly luciferase emits light but does not generate heat, which also causes curiosity because it solves the enzyme-catalyzed reaction. Here we mainly review the structure, mechanism, application, advantages and disadvantages of luciferase.