A Dual‐Color Far‐Red to Near‐Infrared Firefly Luciferin Analogue Designed for Multiparametric Bioluminescence Imaging

Red‐shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red‐shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual‐color, far‐red to near‐infrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far‐red to nIR emission maxima up to λ_max=706 nm with different Fluc mutants. This emission is the most red‐shifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep‐tissue bioluminescence imaging.     

Mechanically Assisted Bioluminescence with Natural Luciferase

Mechanochemical analogues have recently been established for several enzymatic reactions, but they require periodic interruption of the reaction for sampling, dissolution, and (bio)chemical analysis to monitor their progress. By applying a mechanochemical procedure to induce bioluminescence analogous to that used by the marine ostracod Cypridina (Vargula) hilgendorfii, here we demonstrate that the light emitted by a bioluminescent reaction can be used to directly monitor the progress of a mechanoenzymatic reaction without sampling. Mechanical treatment of Cypridina luciferase with luciferin generates bright blue light which can be readily detected and analyzed spectroscopically. This mechanically assisted bioluminescence proceeds through a mechanism identical to that of bioluminescence in solution, but has higher activation energy due to being diffusion‐controlled in the viscous matrix. The results suggest that luciferases could be used as light‐emissive reporters of mechanoenzymatic reactions.     

Progress in the Study of Bioluminescent Earthworms

Even though bioluminescent oligochaetes rarely catch people's eyes due to their secretive lifestyle, glowing earthworms sighting reports have come from different areas on all continents except Antarctica. A major breakthrough in the research of earthworm bioluminescence occurred in the 1960s with the studies of the North American Diplocardia longa. Comparative studies conducted on 13 earthworm species belonging to six genera showed that N‐isovaleryl‐3‐aminopropanal (Diplocardia luciferin) is the common substrate for bioluminescence in all examined species, while luciferases appeared to be responsible for the color of bioluminescence. The second momentous change in the situation has occurred with the discovery in Siberia (Russia) of two unknown luminous enchytraeids. The two bioluminescent systems belong to different types, have different spectral characteristics and localization, and different temperature and pH optima. They are unique, and this fact is confirmed by the negative results of all possible cross‐reactions. The bioluminescent system of Henlea sp. comprises four essential components: luciferase, luciferin, oxygen and calcium ion. For Friderica heliota, the luminescent reaction requires five components: luciferase, luciferin, ATP, magnesium ion and oxygen. Along with luciferin, more than a dozen analogues were isolated from worm biomass. These novel peptide‐like natural compounds represent an unprecedented chemistry found in terrestrial organisms.     

The Sensitized Bioluminescence Mechanism of Bacterial Luciferase

After more than one‐half century of investigations, the mechanism of bioluminescence from the FMNH₂ assisted oxygen oxidation of an aliphatic aldehyde on bacterial luciferase continues to resist elucidation. There are many types of luciferase from species of bioluminescent bacteria originating from both marine and terrestrial habitats. The luciferases all have close sequence homology, and in vitro, a highly efficient light generation is obtained from these natural metabolites as substrates. Sufficient exothermicity equivalent to the energy of a blue photon is available in the chemical oxidation of the aldehyde to the corresponding carboxylic acid, and a luciferase‐bound FMNH‐OOH is a key player. A high energy species, the source of the exothermicity, is unknown except that it is not a luciferin cyclic peroxide, a dioxetanone, as identified in the pathway of the firefly and the marine bioluminescence systems. Besides these natural substrates, variable bioluminescence properties are found using other reactants such as flavin analogs or aldehydes, but results also depend on the luciferase type. Some rationalization of the mechanism has resulted from spatial structure determination, NMR of intermediates and dynamic optical spectroscopy. The overall light path appears to fall into the sensitized class of chemiluminescence mechanism, distinct from the dioxetanone types.     

A new firefly luciferase with bimodal spectrum: identification of structural determinants of spectral pH-sensitivity in firefly luciferases

Fireflies emit flashes in the green-yellow region of the spectrum for the purpose of sexual attraction. The bioluminescence color is determined by the luciferases. It is well known that the in vitro bioluminescence color of firefly luciferases can be shifted toward the red by lower pH and higher temperature; for this reason they are classified as pH-sensitive luciferases. However, the mechanism and structural origin of pH sensitivity in fireflies remains unknown. Here we report the cloning of a new luciferase from the Brazilian twilight active firefly Macrolampis sp2, which displays an unusual bimodal spectrum. The recombinant luciferase displays a sensitive spectrum with the peak at 569 nm and a shoulder in the red region. Comparison of the bioluminescence spectra of Macrolampis, Photinus and Cratomorphus firefly luciferases shows that the distinct colors are determined by the ratio between green and red emitters under luciferase influence. Comparison of Macrolampis luciferase with the highly similar North American Photinus pyralis luciferase (91%) showed few substitutions potentially involved with the higher spectral sensitivity in Macrolampis luciferase. Site-directed mutagenesis showed that the natural substitution E354N determines the appearance of the shoulder in the red region of Macrolampis luciferase bioluminescence spectrum, helping to identify important interactions and residues involved in the pH-sensing mechanism in firefly luciferases.     

Cloning and characterization of an active fragment of luciferase from a luminescent marine alga, Pyrocystis lunula

Two marine dinoflagellates, Lingulodinium polyedrum and Pyrocystis lunula, emit light in a reaction involving the enzymatic oxidation of its tetrapyrrole luciferin by molecular oxygen. The characteristic properties of P. lunula luciferase have not been clarified, whereas L. polyedrum luciferase, which has three active domains, has been characterized. A cloned partial cDNA of the P. lunula luciferase encodes an active fragment corresponding to part of domain 2 and all of domain 3 of L. polyedrum luciferase. The homology of the amino acid sequence between the two luciferases in domain 3 is about 84.3%. A recombinant His-tagged luciferase fragment containing domain 3 (Mr = 46 kDa) catalyzed the light-emitting oxidation of luciferin (lambdamax = 474 nm). This protein was purified by a single affinity-chromatography procedure. The pH-activity profile and the bioluminescence spectrum of the recombinant enzyme having a third domain are almost identical to those of an extract from P. lunula cultured in vitro. The recombinant enzyme is active at pH 8.0, although the recombinant enzyme derived from the second domain of L. polyedrum luciferase is inactive at pH 8.0. Substitution of Glu-201 by histidine in the third domain of P. lunula luciferase showed a decrease of activity above pH 7.0, suggesting that histidine residues could be responsible for pH-sensitivity in dinoflagellate luciferase.     

Bioluminescence is produced from a trapped firefly luciferase conformation predicted by the domain alternation mechanism

According to the domain alternation mechanism and crystal structure evidence, the acyl-CoA synthetases, one of three subgroups of a superfamily of adenylating enzymes, catalyze adenylate- and thioester-forming half-reactions in two different conformations. The enzymes accomplish this by presenting two active sites through an ~140° rotation of the C-domain. The second half-reaction catalyzed by another subgroup, the beetle luciferases, is a mechanistically dissimilar oxidative process that produces bioluminescence. We have demonstrated that a firefly luciferase variant containing cysteine residues at positions 108 and 447 can be intramolecularly cross-linked by 1,2-bis(maleimido)ethane, trapping the enzyme in a C-domain-rotated conformation previously undocumented in the available luciferase crystal structures. The cross-linked luciferase cannot adenylate luciferin but is nearly fully capable of bioluminescence with synthetic luciferyl adenylate because it retains the ability to carry out the oxidative half-reaction. The cross-linked luciferase is apparently trapped in a conformation similar to those adopted by acyl-CoA synthetases as they convert acyl adenylates into the corresponding CoA thioesters.     

Luciferin Regeneration in Firefly Bioluminescence via Proton-Transfer-Facilitated Hydrolysis,Condensation and Chiral Inversion

Firefly bioluminescence is produced via luciferin enzymatic reactions in luciferase. Luciferin has to be unceasingly replenished to maintain bioluminescence. How is the luciferin reproduced after it has been exhausted? In the early 1970s, Okada proposed the hypothesis that the oxyluciferin produced by the previous bioluminescent reaction could be converted into new luciferin for the next bioluminescent reaction. To some extent, this hypothesis was evidenced by several detected intermediates. However, the detailed process and mechanism of luciferin regeneration remained largely unknown. For the first time, we investigated the entire process of luciferin regeneration in firefly bioluminescence by density functional theory calculations. This theoretical study suggests that luciferin regeneration consists of three sequential steps: the oxyluciferin produced from the last bioluminescent reaction generates 2-cyano-6-hydroxybenzothiazole (CHBT) in the luciferin regenerating enzyme (LRE) via a hydrolysis reaction; CHBT combines with L-cysteine in vivo to form L-luciferin via a condensation reaction; and L-luciferin inverts into D-luciferin in luciferase and thioesterase. The presently proposed mechanism not only supports the sporadic evidence from previous experiments but also clearly describes the complete process of luciferin regeneration. This work is of great significance for understanding the long-term flashing of fireflies without an in vitro energy supply.     

Theoretical Study of Dinoflagellate Bioluminescence

Dinoflagellates are the most ubiquitous luminescent protists in the marine environment and have drawn much attention for their crucial roles in marine ecosystems. Dinoflagellate bioluminescence has been applied in underwater target detection. The luminescent system of dinoflagellates is a typical luciferin–luciferase one. However, the excited‐state oxyluciferin is not the light emitter of dinoflagellate bioluminescence as in most luciferin–luciferase bioluminescent organisms. The oxyluciferin of bioluminescent dinoflagellates is not fluorescent, whereas its luciferin emits bright fluorescence with similar wavelength of the bioluminescence. What is the light emitter of dinoflagellate bioluminescence and what is the chemical process of the light emission like? These questions have not been answered by the limited experimental evidence so far. In this study, for the first time, the density functional calculation is employed to investigate the geometries and properties of luciferin and oxyluciferin of bioluminescent dinoflagellate. The calculated results agree with the experimental observations and indicate the luciferin or its analogue, rather than oxyluciferin, is the bioluminophore of dinoflagellate bioluminescence. A rough mechanism involving energy transfer is proposed for dinoflagellate bioluminescence.     

Firefly Luciferase Bioluminescence as a Tool for Searching Magnetic Isotope Effects in ATP-Dependent Enzyme Reactions

Cells and tissues are composed from atoms of chemical elements, some of which have two kinds of stable isotopes, magnetic and nonmagnetic ones. Not long ago, magnetic isotope effects (MIEs) have been discovered in experiments with cells enriched with magnetic or nonmagnetic isotopes of magnesium. These MIEs can stem from higher efficiency of the enzymes of bioenergetics in the cells enriched with magnetic magnesium isotope. In the studies of MIEs in biological systems, it is needed to monitor the ATP concentrations as the major energy source in cells. The most sensitive and rapid method of the ATP measurements is based on the use of the firefly luciferase–luciferin system. Since luciferase is the ATP-dependent enzyme and activated by Mg-ions, it is necessary to elucidate whether this enzyme is sensitive to magnetic field of the magnesium isotope’s nuclear spin. Herein we present the results of studying the effects of different isotopes of magnesium, magnetic ²⁵Mg and nonmagnetic ²⁴Mg and ²⁶Mg, on bioluminescence spectra and enzymatic activity of firefly luciferase. It was shown, that neither kinetics of the bioluminescence signal nor the bioluminescence spectra manifest any statistically significant dependence on the type of magnesium isotope. So, no MIEs have been revealed in the luciferase-catalyzed oxidation of luciferin. It means that firefly luciferase bioluminescence can serve as the tool for search and studies of magnetic isotope effects in ATP-dependent enzyme reactions in biological systems, including the enzymatic synthesis and hydrolysis of ATP.     

Firefly Luciferase‐based Fusion Proteins and their Applications in Bioanalysis

Firefly luciferase is widely used in molecular biology and bioanalytical systems as a reporter molecule due to the high quantum yield of the bioluminescence, availability of stable mutant forms of the enzyme with prescribed spectral characteristics and abundance of bacterial expression systems suitable for production of recombinant proteins in limitless quantities. In this review, we described fusion proteins of luciferase with biotin‐binding domain and streptavidin, with proteins A and G, antibodies, with DNA‐ and RNA‐binding proteins, as well as fusion proteins designed for BRET systems. The firefly luciferase‐based fusion proteins are represented as an effective tool for the development of different bioanalytical systems such as (1) systems in which luciferase is attached to the surface of the target and the bioluminescence signal is detected from the specific complexes formed; (2) BRET‐based systems, in which the specific interaction induces changes in the bioluminescence spectrum; and (3) systems that use modified or split luciferases, in which the luciferase activity changes under the action of the analyte. All these systems have wide application in biochemical analysis of physiologically important compounds, for the detection of pathogenic bacteria and viruses, for evaluation of protein–protein interactions, assaying of metabolites involved in cell communication and cell signaling.     

Yellow-green and red firefly bioluminescence from 5,5-dimethyloxyluciferin

Beetle luciferases (including those of the firefly) use the same luciferin substrate to naturally display light ranging in color from green (lambda(max) similar 530 nm) to red (lambda(max) similar 635 nm). The original mechanism of bioluminescence color determination advanced by White and co-workers was based on the concept that the keto and enol tautomers of the emitter oxyluciferin produce red and green light, respectively. Alternatively, McCapra proposed that color variation is associated with conformations of the keto form of excited-state oxyluciferin. We have prepared the adenylate of D-5,5-dimethylluciferin and shown that it is transformed into the putative emitter 5,5-dimethyloxyluciferin in bioluminescence reactions catalyzed by luciferases from Photinus pyralis and the green-emitting click beetle. 5,5-Dimethyloxyluciferin is constrained to exist in the keto form and fluoresces in the red. However, bioluminescence spectra revealed that green light emission was produced by the firefly enzyme and red light was observed with the click beetle protein. These results, augmented with steady-state kinetic studies, may be taken as the first experimental support for McCapra's mechanism of firefly bioluminescence color or any other proposal that requires only a single keto form of oxyluciferin.     

Limited Proteolysis of Luciferase as a Reporter in Nanosystem Biology: A Comparative Study

Firefly luciferase is a 62 kDa protein that produces a flash of light on the oxidation of luciferin in the presence of ATP, Oxygen and Mg²⁺. Luciferase has a broad range of applications for analytical purposes and in vivo imaging for nanosystem biology studies. However, the enzyme is highly susceptible to proteolytic degradation that reduces its half-life. Rate of proteolytic digestion between two members of luciferase family ( Photinus pyralis and Lampyris turkestanicus ) is compared. Proteolytic sensitivity of L. turkestanicus luciferase was found to be more than P. pyralis luciferase, due to higher rate of hydrolysis under identical conditions. Both luciferases showed more sensitivity to chymotrypsin than trypsin with different digestion pattern. Digestion of P. pyralis by trypsin produced some fragments which were found to be more resistant to further degradation, whereas in L. turkestanicus initial fragments subdigested by trypsin, like chymotrypsin effect on both luciferases. Furthermore, both luciferases become increasingly labile to proteolysis as the temperature increases. The rate of inactivation and the rate of degradation between luciferases were different in a specific time of incubation. Appearance of similar bands for both luciferases confirmed exposure of specific regions, in spite of structural differences.     

Expedient synthesis of electronically modified luciferins for bioluminescence imaging

Bioluminescence imaging with luciferase enzymes requires access to light-emitting, small-molecule luciferins. Here, we describe a rapid method to synthesize d-luciferin, the substrate for firefly luciferase (Fluc), along with a novel set of electronically modified analogues. Our procedure utilizes a relatively rare, but synthetically useful dithiazolium reagent to generate heteroaromatic scaffolds in a divergent fashion. Two of the luciferin analogues produced with this approach emit light with Fluc in vitro and in live cells. Collectively, our work increases the number of substrates that can be used for bioluminescence imaging and provides a general strategy for synthesizing new collections of luciferins.     

Directed Improvement of Luciferin Regenerating Enzyme Binding Properties: Implication of Some Conserved Residues in Luciferin‐Binding Domain

Luciferin regenerating enzyme (LRE) contributes to in vitro recycling of d ‐luciferin to produce persistent and longer light emission by luciferase. Luciferin binding domains I and II among LREs regarded as potential candidates for luciferin‐binding sites. In this study, for the first time, amino acids T69, G75 and K77 located at luciferin binding domain I of LRE from L. turkestanicus (T‐LRE) substituted by using site‐directed mutagenesis. Single mutant T⁶⁹R increased luciferase light output more than two‐fold over a longer time in comparison with a wild‐type and other mutants of T‐LRE. Nevertheless, double mutant (K⁷⁷E/T⁶⁹R) increased the amount of bioluminescent signal more than two‐fold over a short time. In addition, G⁷⁵E, K⁷⁷E and G⁷⁵E/T⁶⁹R mutants did not improve luciferin–luciferase in vitro bioluminescence. Based on our results, addition of K⁷⁷E/G⁷⁵E and K⁷⁷E/G⁷⁵E/T⁶⁹R mutants caused intermediate changes in bioluminescence from in vitro luciferin–luciferase reaction. These findings indicated that the amino acids in question are possible to be located within T‐LRE active site. It may also be suggested that substituted Arg69 (Arg218) plays an important role in luciferin binding and the existence of Gly75 as well as Lys77 is essential for T‐LRE which has already evolved to have different functions in nature.     

The influence of the loop between residues 223-235 in beetle luciferase bioluminescence spectra: a solvent gate for the active site of pH-sensitive luciferases

Beetle luciferases emit a wide range of bioluminescence colors, ranging from green to red. Firefly luciferases can shift the spectrum to red in response to pH and temperature changes, whereas click beetle and railroadworm luciferases do not. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. Through comparative site-directed mutagenesis and modeling studies, using the pH-sensitive luciferases (Macrolampis and Cratomorphus distinctus fireflies) and the pH-insensitive luciferases (Pyrearinus termitilluminans, Phrixotrix viviani and Phrixotrix hirtus) cloned by our group, here we show that substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). The substitutions at positions 227, 228 and 229 (P. pyralis sequence) cause dramatic redshift and temporal shift in both groups of luciferases, indicating their involvement in labile interactions. Modeling studies showed that the residues Y227 and N229 are buried in the protein core, fixing the loop to other structural elements participating at the bottom of the luciferin binding site. Changes in pH and temperature (in firefly luciferases), as well as point mutations in this loop, may disrupt the interactions of these structural elements exposing the active site and modulating bioluminescence colors.     

Synthesis of Firefly Luciferin Analogues and Evaluation of the Luminescent Properties

Five new firefly luciferin ( 1 ) analogues were synthesized and their light emission properties were examined. Modifications of the thiazoline moiety in 1 were employed to produce analogues containing acyclic amino acid side chains ( 2 – 4 ) and heterocyclic rings derived from amino acids ( 5 and 6 ) linked to the benzothiazole moiety. Although methyl esters of all of the synthetic derivatives exhibited chemiluminescence activity, only carboluciferin ( 6 ), possessing a pyrroline‐substituted benzothiazole structure, had bioluminescence (BL) activity (λ_max=547 nm). Results of bioluminescence studies with AMP‐carboluciferin (AMP=adenosine monophosphate) and AMP‐firefly luciferin showed that the nature of the thiazoline mimicking moiety affected the adenylation step of the luciferin–luciferase reaction required for production of potent BL. In addition, BL of 6 in living mice differed from that of 1 in that its luminescence decay rate was slower.     

Bioluminescence activity of Latia luciferin analogues: replacement of the 2,6,6-trimethylcyclohexene ring onto the methyl-substituted phenyl groups

A series of Latia luciferin analogues having methyl-substituted phenyl groups instead of the natural 2,6,6-trimethylhexene ring was synthesized and their bioluminescence activity were measured. The Latia luciferase was found to be able to moderately recognize the appropriately methyl-substituted phenyl analogues with the same light production kinetics as that of natural luciferin.     

CONSTRUCTION AND CHARACTERIZATION OF HYBRID LUCIFERASES CODED BY lux GENES FROM Xenorhabdus luminescens AND Vibrio fischeri

Abstract— Molecular cloning techniques were employed to obtain hybrid luciferases with their α and β subunits encoded by lux A and lux B genes, respectively, from Xenorhabdus luminescens strain HW or Vibrio fischeri. Although the two wild-type luminous bacteria are phylogenetically diverged, the hybrid luciferase Xf comprising an α from V. fischeri and a β from X. luminescens HW were both functional in bioluminescence. Their general kinetic properties were close to the wild type enzymes from which the α subunit was derived. The X. luminescens HW enzyme is distinct in having a high optimal temperature for in vitro bioluminescence, a high thermal stability and a sensitivity to aldehyde substrate inhibition. Comparisons of the Xf and VI hybrid luciferases with the two wild-type enzymes indicated that these unusual properties of the X. luminescens HW luciferase originated primarily from the α subunit.     

Identification of mutant firefly luciferases that efficiently utilize aminoluciferins

Firefly luciferase-catalyzed light emission from D-luciferin is widely used as a reporter of gene expression and enzymatic activity both in vitro and in vivo. Despite the power of bioluminescence for imaging and drug discovery, light emission from firefly luciferase is fundamentally limited by the physical properties of the D-luciferin substrate. We and others have synthesized aminoluciferin analogs that exhibit light emission at longer wavelengths than D-luciferin and have increased affinity for luciferase. However, although these substrates can emit an intense initial burst of light that approaches that of D-luciferin, this is followed by much lower levels of sustained light output. Here we describe the creation of mutant luciferases that yield improved sustained light emission with aminoluciferins in both lysed and live mammalian cells, allowing the use of aminoluciferins for cell-based bioluminescence experiments.