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.     

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.     

Quantum yields and quantitative spectra of firefly bioluminescence with various bivalent metal ions

We measured quantitative spectra of firefly (Photinus pyralis) bioluminescence in the presence of Zn²⁺ and other bivalent metal ions to investigate the effects of these metal ions on luciferin‐luciferase reaction. We studied the dependence of the quantum yield and spectrum on quantity and kind of bivalent metal ions. Adding various amounts of Mg²⁺, Mn²⁺ and Ca²⁺ produced virtually no change in the quantum yields or the spectra of bioluminescence. In contrast, increasing amounts of ions such as Zn²⁺ and Cd²⁺ decreased quantum yields and changed the bioluminescence color from yellow‐green to red. Quantitative analysis showed that the sensitivities of the quantum yield and color to various metal ions were in the order of Hg²⁺>Zn²⁺, Cd²⁺>Ni²⁺, Co²⁺, Fe²⁺≫Mg²⁺, Mn²⁺, Ca²⁺. We propose that the changes in quantum yield and spectrum caused by the metal ions are due to their effect on luciferase that surrounds oxyluciferin during its radioactive decay. We also found that having more metal ions accelerated bioluminescence reactions. The sensitivity of the reaction rate had no correlation with those of the quantum yield and spectrum.     

BIOLUMINESCENCE OF BRAZILIAN FIREFLIES (COLEOPTERA: LAMPYRIDAE): SPECTRAL DISTRIBUTION and pH EFFECT ON LUCIFERASE-ELICITED COLORS. COMPARISON WITH ELATERID and PHENGODID LUCIFERASES

Twenty-five Brazilian species (nine genera: Phorinus, Photinoides, Macrolampis, Aspisoma, Cratomorphus, Amydetes, Photuris, Bicellonychia, Pyrogaster) of adult fireflies were found to emit light in vivo in the green-yellow range (Λ_max=548–573 nm) of the spectrum, more frequently near the green region, in contrast with North-American species, which predominantly emit yellow light. Distinct ecological contexts where these species evolved, such as the habitat (open field vs forests) and the duration of twilight, are discussed as possible factors responsible for these differences. Except for Photuris and Bicellonychia spp., the in vivo and in vitro bioluminescence spectra for various species of a given genus agree within ±5 nm. Lowering the pH caused the typical red shift in the in vitro bioluminescence spectrum from lampyrid luciferases (six species), which has been interpreted as due to the presence of a basic residue in the enzyme active site catalyzing fast enolization of the initially formed excited keto-oxyluciferin (red emitter) to the excited enol form (yellow-green emitter). The in vitro bioluminescence colors obtained from larval or adult elaterid (five species) and phengodid (three species) luciferases studied here, spanning the green-red region, do not respond to pH changes. This could indicate either the absence of the neighboring basic center (in red-emitting luciferases) or the presence of a non-pH affected proximal basic residue in the active site of the luciferase (in yellow-green-emitting luciferases).     

Recombinant firefly luciferase in Escherichia coli

The authentic recombinant luciferase, the luciferase with the structure similar to that of the native protein, was obtained using random mutagenesis, and its properties were studied in comparison with several fusion proteins. Thermoinactivation curves of the recombinant luciferases within the 10–50°C temperature interval showed that thermoinactivation involves reversible and irreversible steps. Immobilization of the recombinant Luciola mingrelica and Photinus pyralis firefly luciferases on BrCN-activated sepharose was carried out. Immobilization resulted in the preparation of enzymes with high catalytic activity. Physicochemical properties and analytical characteristics of the immobilized recombinant and native luciferases were studied. The catalytic properties of the immobilized recombinant L. mingrelica luciferase were close to those of the native luciferase but the former enzyme appeared to be significantly more stable. The immobilized recombinant luciferases can be used for ATP assay within 0.01–10000 nM range.     

Bioluminescence color determinants of Phrixothrix railroad-worm luciferases: chimeric luciferases, site-directed mutagenesis of Arg 215 and guanidine effect

Chimeric proteins were produced using the green light-emitting luciferase of Phrixothrix vivianii (PxGr: lambda max = 548 nm) and the red light-emitting luciferase of Phrixothrix hirtus (PxRe: lambda max = 623 nm). Constructs containing residues 1-344 of the red light-emitting luciferase with residues 345-545 of the green light emitting one emitted red light (PxReGr; lambda max = 613 nm), while the reverse emitted green light (PxGrRe; lambda max = 552 nm). From these results we conclude that the region 1-344 determines the color of bioluminescence (BL) in railroad-worm luciferases, and that residues above 344 are not involved. The substitution R215S in the green light-emitting luciferase (PxGr) resulted in a approximately 40 nm redshift on the BL spectrum (lambda max = 585 nm) and an associated decrease of activity, whereas the same mutation in PxRe luciferase had little effect. Guanidine was shown to cause blueshifts in the BL spectra and stimulate the activity of the red-emitting luciferases (from lambda max = 623 to lambda max = 600 nm) and in PxGr R215S (from lambda max = 585 to lambda max = 560 nm) mutant luciferase, but not in the green-emitting luciferases, suggesting that guanidine can simulate positively charged residues involved in BL color determination.     

Quantum Yields and Kinetics of the Firefly Bioluminescence Reaction of Beetle Luciferases

Quantum yields of firefly bioluminescence reactions were determined for beetle luciferases from the three main families of luminous beetles emitting different bioluminescence colors. Quantum yield (QY) was significantly correlated with luminescence spectrum. The green light-emitting luciferase of the Brazilian click beetle, Pyrearinus termitilluminans, whose luminescence spectrum had the shortest peak wavelength of all the luciferases investigated, had the highest QY (0.61). Mutant analyses of active site-substituted Pyrocoelia miyako luciferases showed that, although k_cat was decreased by the mutations, the QY was not significantly affected.     

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.     

Fragment molecular orbital investigation of the role of AMP protonation in firefly luciferase pH-sensitivity

Firefly bioluminescence displays a sensitivity to pH changes through an alteration of the energy of the emitted photon leading to yellow-green light above ～pH 6.5 and red light below this value. Calculations using the fragment molecular orbital method have been performed on the active site of the luciferase enzyme from the Japanese firefly Luciola cruciata in order to investigate both the importance of different protonation states and tautomeric forms of the lumophore, oxyluciferin, and the role played by protonation of the active site AMP molecule. The results suggest that whilst an equilibrium between several protonation/tautomeric states of oxyluciferin is possible, a single oxyluciferin species (the phenolate-keto form) may be mostly responsible for both emission colours, with changes in polarization by the active site caused by protonation of the AMP molecule playing an important role in mediating the pH-dependent shift.     

The influence of Ala243 (Gly247), Arg215 and Thr226 (Asn230) on the bioluminescence spectra and pH-sensitivity of railroad worm,click beetle and firefly luciferases

Among beetle luciferases, the pH-sensitive firefly luciferases have been studied extensively. Much less is known about pH-insensitive luciferases, which include click beetle and railroad worm luciferases. Previously, we found that the residues R215 and T226 (N230) are important for green light emission. Here we show that the conserved residue A243 in pH-insensitive luciferases and the corresponding G247 in pH-sensitive luciferases affect the emission spectrum and influence pH-sensitivity. In contrast to railroad worm green light-emitting (PxvGR) and firefly luciferases, the substitution of R215 in Pyrearinus termitilluminans click beetle luciferase (Pte) had no effect on the spectrum, showing that R215 is not essential for green light emission in all beetle luciferases. A homology-based model of Pte luciferase shows that R215 and T226 are close enough to interact. To investigate if there was an interaction between these conserved residues, double mutants were constructed. The double substitution R215S/T226N in Pte luciferase abolished the activity. In PxvGR luciferase the same double mutant resulted in a redshift (lambda(max) = 595 nm), whose magnitude was lower than the value expected for an additive effect. These results suggest that the effects of R215S and T226N are partially interdependent. The double substitution T226N/A243G had an additive redshift effect on the spectrum of PxvGR luciferase, whereas it had a smaller effect on the spectrum of Pte luciferase. Altogether, these results suggest that the above substitutions have different effects on the active site of click beetle and railroad worm luciferases.     

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-BASED PURIFICATION OF FIREFLY LUCIFERASES

Abstract— A simple procedure is described for purifying luciferases from firefly lanterns in greater than 50% yield. The key step in the method is high-performance liquid chromatography (HPLC) using a Bio-Gel TSK DEAE-5PW analytical (75 times 7.5 mm) anion exchange column. In separate experiments, partially purified protein solutions obtained from Photinus pyralis and Photinus macder-motti lanterns by solubilization, ammonium sulfate fractionation and Sephadex G-25 gel filtration were chromatographed. Luciferases from both species had relative molecular masses (M_g) equal to 61 ± 2 times 10³ and were strongly reactive to anti-P. pyralis luciferase antibody. Isoelectric focusing (IEF) experiments, HPLC retention times ( R _t), and bioluminescence emission spectra demonstrated that the luciferases from the different Photinus species were very similar, but not identical. The purification procedures described are most suitable for the isolation of 2–10 mg of protein; however, the use of a preparative column should enable the convenient isolation of larger amounts of protein. Also, this method should be readily adaptable to the isolation of luciferases from additional genera and species of fireflies.     

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.     

CLONING, EXPRESSION and SEQUENCE ANALYSIS OF cDNA FOR THE LUCIFERASES FROM THE JAPANESE FIREFLIES, Pyrocoelia tniyako AND Hotaria parvula

Abstract— Cloning and sequence analysis of cDNA for the luciferases of Pyrocoelia miyako and Hotaria parvula were carried out (GenBank accession numbers L39928 and L39929, respectively). The amino acid sequence, deduced from the nucleotide sequence, showed P. miyako luciferase to consist of 548 amino acid residues with a molecular weight of 60955, while the luciferase of H. parvula consisted of 548 amino acid residues with a molecular weight of 60 364. Pyrocoelia miyako luciferase showed 82.1 % homology with the luciferase of Photinus pyralis and less than 70% homology with other firefly luciferases, whereas H. parvula luciferase showed 98%, 82.5% and 81.2% homology with the luciferases of Luciola mingrelica, Luciola lateralis and Luciola cruciata, respectively. Two regions in the enzymes were found to be highly conserved. The amino acid sequences were used to construct a phylogenetic tree, which showed that the fireflies could be divided into two groups.     

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.     

Creation of a thermostable firefly luciferase with pH-insensitive luminescent color

The thermal instability and pH-sensitive spectral property of firefly luciferase have hampered its use as a sensitive multicolor luminescent label or bioluminescent resonance energy transfer donor. With the intention of improving the thermostability of a previously found firefly Hotaria parvula luciferase mutant with minor pH-sensitive spectral change (V368A), further mutation (E356R) was introduced by taking a reportedly stabilized mutant of Photinus pyralis luciferase into account. The double mutant E356R/V368A showed significantly improved thermostability because > 90% activity remained after incubation for 1 h at 45 degrees C, with its specific activity being maintained. Unlike the wild type or V368A, E356R/V368A showed no change in the emission maximum of 568 nm even at pH 6.3, also implying a mutual relationship between thermostability and the proportion of yellow-green luminescent peak under acidic condition.     

Toward bioluminescence in the near-infrared region: Tuning the emission wavelength of firefly luciferin analogues by allyl substitution

The synthesis and bioluminescence of allyl-substituted luciferin derivatives as substrates for firefly luciferase are reported. The allylation of luciferins induced bathochromic shift (15–40?nm) of the bioluminescence emission. Upon combination with other chemical modifications for bioluminescence wavelength tuning, novel red emitting luciferin analogues were obtained with emission maxima at 685 and 690?nm.     

Quantum yield improvement of red-light-emitting firefly luciferin analogues for in vivo bioluminescence imaging

The dimethylamino group of AkaLumine ((4S)-2-[(1E,3E)-4-[4-(dimethylamino)phenyl]-1,3-butadien-1-yl]-4,5-dihydro-4-thiazolecarboxylic acid), a red-light-emitting firefly luciferin analogue, was replaced by cyclic amino groups (1-pyrrolidinyl, 1-piperidino, 1-azepanyl, and 4-morpholino) to give AkaLumine analogues exhibiting desirable bioluminescence with emission maxima in the red region (656–667 nm). In particular, a bioluminescence reaction of 1-pyrrolidinyl analogue with a recombinant Photinus pyralis luciferase showed a higher quantum yield than that with AkaLumine, giving an improved bioluminescence intensity. The 1-pyrrolidinyl analogue also showed the strongest luminescence in whole-body luciferase-expressing mice among the analogues, indicating that a quantum yield improvement of a luciferin analogue is effective to increase bioluminescence imaging intensity.     

Theoretical modulation of the color of light emitted by firefly oxyluciferin

One of the major mysteries regarding firefly bioluminescence is its pH-dependent multicolor variation. At basic pH, the emission is on the yellow-green region, whereas at acid pH, the light emission is observed on the red region of the visible spectrum. Theoretical calculations using density functional theory, molecular mechanics, and semiempirical methods were made to investigate the effect exerted by intermolecular forces on light emission, and their modulation by polarity, and the differences in the conformation of the active site at basic and acid pH. Red emission is achieved by the weakening of the interactions of the emitter with ionic and hydrophobic molecules, by the polarization of the benzothiazole microenvironment, by ionization of the enzyme-emitter complex and by changes of the hydrogen bond network. Arg220, Glu346, Ala350, Leu344 and adenosine-5'-monophosphate have blue-shifting effects, while His247, Phe249, Gly341, Thr253, and Ile288 exert a redshifting one.