Quenching of tryptophan fluorescence of firefly luciferase by substrates

The interaction of firefly luciferase with substrates (luciferin and MgATP) by steady-state and time-resolved fluorescence is studied. The efficient quenching of tryptophan fluorescence of the active enzyme takes place upon its binding with substrates. In the presence of ATP the quenching is of dynamic type and is caused by structural changes in the protein molecule upon ATP binding. A model is proposed in which the complex has smaller fluorescence quantum yield than the free enzyme because of partial quenching of tryptophan fluorescence by the new microenvironment. Quenching of tryptophan fluorescence by luciferin due to the efficient energy transfer from tryptophan to luciferin is discussed. The calculated distance between Trp-419 and luciferin for the L. mingrelica luciferase in the enzyme-substrate complex is less than 12 A.     

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.     

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.     

Larval Tenebrio molitor (Coleoptera: Tenebrionidae) Fat Body Extracts Catalyze Firefly D-Luciferin- and ATP-Dependent Chemiluminescence: A Luciferase-like Enzyme*

Ultraweak light emission was detected upon injection of firefly luciferin into live Tenebrio larvae. A chemilumi-nescent enzymatic activity dependent on molecular oxygen, D-luciferin and MgATP was then isolated from larval fat body extracts by precipitation with 70% ammonium sulfate. D-Luciferin and ATP can be replaced by luciferyl-adenylate. Pyrophosphate is a main product from the chemiluminescent reaction. The in vitro chemiluminescence intensity was not affected by peroxidase inhibitors such as N₃^?_- (0.5 mM) and CN^? (1 mM), attesting to its nonperoxidatic nature but was strongly inhibited by AMP (1 mM), luciferin 6′-ethyl ether (1 mM) and sodium pyrophosphate (2 mM), well-known firefly lucifer-ase inhibitors. Some physical-chemical properties of this enzymatic activity were similar to those of firefly lucif-erase (K_MATP = 195 μM; K_0.5 luciferin - 0.8 mM; optimum pH 8.5; δ_max= 610 nm at pH 8.5; firefly lucifer-ase: δ_max= 565 nm at pH 8.0 and 619 mm at pH 6.0), but the chemiluminescence was not affected by addition of polyclonal antibodies raised against Photinus pyralis luciferase. These data suggest that this chemiluminescence results from a ligase with luciferase activity.     

Carboxymethyl poly(L‐histidine) as a new pH‐sensitive polypeptide at endosomal/lysosomal pH

A carboxymethyl poly(L ‐histidine) has been synthesized as a new pH‐sensitive polypeptide at endosomal/lysosomal pH. Because of its poor water solubility at physiological pH, an application of poly(L ‐histidine) with a pK_a around 6.0 has been limited in spite of the native possession of the pH‐dependent property change at endosomal pH. Although the unmodified poly(L ‐histidine) suddenly precipitates out of the aqueous medium above pH 6.0 as the result of the deprotonation of the imidazole groups, the water solubility of the resulting carboxymethyl poly(L ‐histidine) has been improved at physiological pH. A solution turbidity measurement proved that no significant effect on a rapid aggregate formation or phase separation of serum proteins is induced by carboxymethyl poly(L ‐histidine). Hemolysis assay showed that the carboxymethyl poly(L ‐histidine) enhances membrane disruptive ability at endosomal/lysosomal pH. The cellular uptake of luciferase in the presence of the carboxymethyl poly(L ‐histidine) increases intracellular luciferase activity, which suggests that the carboxymethyl poly(L ‐histidine) makes the luciferase escape from lysosomal degradation. The carboxymethyl poly(L ‐histidine) would be the fundamental compound for designing various drug carriers with the pH sensitivity at endosomal/lysosomal pH. Copyright © 2007 John Wiley & Sons, Ltd.     

Firefly Luciferase Mutants Allow Substrate‐Selective Bioluminescence Imaging in the Mouse Brain

Bioluminescence imaging is a powerful approach for visualizing specific events occurring inside live mice. Animals can be made to glow in response to the expression of a gene, the activity of an enzyme, or the growth of a tumor. But bioluminescence requires the interaction of a luciferase enzyme with a small‐molecule luciferin, and its scope has been limited by the mere handful of natural combinations. Herein, we show that mutants of firefly luciferase can discriminate between natural and synthetic substrates in the brains of live mice. When using adeno‐associated viral (AAV) vectors to express luciferases in the brain, we found that mutant luciferases that are inactive or weakly active with d ‐luciferin can light up brightly when treated with the aminoluciferins CycLuc1 and CycLuc2 or their respective FAAH‐sensitive luciferin amides. Further development of selective luciferases promises to expand the power of bioluminescence and allow multiple events to be imaged in the same live animal.     

Mechanisms in the quantum yield of Cypridina bioluminescence

Abstract— –The influence of temperature, pH, salts, and reactant concentrations on the biolumin-escent oxidation of Cypridina luciferin catalyzed by Cypridina luciferase indicates a highest quantum yield φ (einsteins per mole of luciferin oxidized) of 0.31 in H₂O, or 0.33 in 99% D₂O. With the aid of data on fluorescence of the light-emitting oxyluciferin-luciferase complex, and of oxyluciferin in diglyme, partial explanations are suggested for the observed variations in φ, including the relatively low φ, of 0.03 for chemiluminescence of luciferin in organic solvents, wherein a different pathway of luciferin degradation, as indicated by chromatographic evidence, results in much less population of the excited state.     

Syntheses and evaluation of the bioluminescent activity of (S)-Cypridina luciferin and its analogs

Cypridina luciferin is the substrate in the bioluminescence of a luminous ostracod Cypridina (Vargula) hilgendorfii. Cypridina luciferin contains a chiral center in the sec-butyl moiety. Here, we report a convenient method for the preparation of (S)-Cypridina luciferin by the condensation of (S)-1,1-diethoxy-3-methylpentan-2-one with ethioluciferin. The light yield of the synthesized (S)-luciferin in the presence of Cypridina luciferase was about 1.7 times as active as that of racemic form. Furthermore, several luciferin analogs prepared by the same condensation with different α-ketoacetal derivatives showed moderate light yield with Cypridina luciferase. These readily available Cypridina luciferin and analogs are applicable to the bioluminescent detection of Cypridina 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.     

Cloning of the Orange Light‐Producing Luciferase from Photinus scintillans—A New Proposal on how Bioluminescence Color is Determined

Unlike the enchanting yellow‐green flashes of light produced on warm summer evenings by Photinus pyralis, the most common firefly species in North America, the orange lights of Photinus scintillans are infrequently observed. These Photinus species, and likely all bioluminescent beetles, use the same substrates beetle luciferin, ATP and oxygen to produce light. It is the structure of the particular luciferase enzyme that is the key to determining the color of the emitted light. We report here the molecular cloning of the P. scintillans luc gene and the expression and characterization of the corresponding novel recombinant luciferase enzyme. A comparison of the amino acid sequence with that of the highly similar P. pyralis enzyme and subsequent mutagenesis studies revealed that the single conservative amino acid change tyrosine to phenylalanine at position 255 accounted for the entire emission color difference. Additional mutagenesis and crystallographic studies were performed on a H‐bond network, which includes the position 255 residue and five other stringently conserved beetle luciferase residues, that is proximal to the substrate/emitter binding site. The results are interpreted in the context of a speculative proposal that this network is key to the understanding of bioluminescence color determination.     

Purification and partial spectral characterization of a novel luciferin from the luminous enchytraeid Fridericia heliota

A homogeneous luciferin preparation has been obtained from the luminous soil enchytraeid Fridericia heliota, which has an ATP-dependent luminescent system. A procedure for luciferin purification without losing fractions of active luciferase has been developed. The luciferin specific activity is 4000 times increased; its UV absorption spectrum maximum is 294 nm with a local minimum at 262 nm. The luciferin of the enchytraeid F. heliota is significantly different from firefly luciferin, whose luminescent reaction also requires ATP, and it also appears to have no similarities to other known luciferins.     

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.     

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.     

Luciferins, bioluminescent substances

The bioluminescence of the American firefly Photinus is due to the reaction of 2-(6-hydroxybenzothiazol-2-yl)-Δ²-1,3-thiazoline-4-carboxylic acid (“firefly luciferin”) with the enzyme luciferase in the presence of ATP and magnesium ion. In the crustacean Cypridina, on the other hand, the bioluminescence is due to the reaction of a luciferase with 8-(3-guanidinopropyl)-6-indol-3-yl-2-(1-methylpropyl)-3,7-dihydroimidazo[1,2-a]-pyrazin-3-one (“Cypridina luciferin”). The luciferin in Latia is 1,3,3-trimethyl-2-(4-formyloxy-3-methyl-3-butenyl)-1-cyclohexene and that in Renilla is a tryptamine derivative that has not yet been accurately identified; the luciferins of other luminescent organisms are not yet known. A review is given of the investigations which have been carried out on the above luciferins and the course of the luciferin-luciferase reaction is examined. Numerous spectral data obtained during the examination of these compounds are included in the text.     

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.     

Structure of cypridina biluciferyl,a dimer of cypridina luciferyl radical having bioluminescent activity

Structure of $\text{Cypridina}$ biluciferyl (luciferyl radical dimer), which is produced by chemical oxidation of $\text{C}$. luciferin with such as ferricyanide, was determined to be the symmetric 5,5′-dimer of $\text{C}$. luciferin. It gives light in the presence of $\text{C}$. luciferase, although the bioluminescent rate is very low. We suggest that the biluciferyl is an intermediate in the oxidation of the luciferin to $\text{C}$. luciferinol.     

Bioluminescence of monolayers of firefly luciferase immobilized on graphite

We report on the immobilization of the firefly protein luciferase on the hydrophobic surface of graphite. Observation by liquid-phase atomic force microscopy of islands with a height consistent with the size of a single molecule confirmed that the protein was contained within a monomolecular layer. The enzyme activity was assayed by single-photon counting of the bioluminescence, which is the catalytic product of luciferase. Attachment to the surface modified the efficiency of the enzyme, but the introduction of the substrates luciferin and ATP resulted in the reactivation of the enzyme. The functionalized graphite surface was employed as a cathode in a bioelectrochemical cell. This demonstrated that the electric field caused a substantial loss of enzyme catalytic activity.