Role of Histidine 310 in Amydetes vivianii firefly luciferase pH and metal sensitivities and improvement of its color tuning properties.

Firefly luciferases emit yellow-green light and are pH-sensitive, changing the bioluminescence color to red in the presence of heavy metals, acidic pH and high temperatures. These pH and metal-sensitivities have been recently harnessed for intracellular pH indication and toxic metal biosensing. However, whereas the structure of the pH sensor and the metal binding site, which consists mainly of two salt bridges that close the active site (E311/R337 and H310/E354), has been identified, the specific role of residue H310 in pH and metal sensing is still under debate. The Amydetes vivianii firefly luciferase has one of the lowest pH sensitivities among the group of pH-sensitive firefly luciferases, displaying high bioluminescent activity and special spectral selectivity for cadmium and mercury, which makes it a promising analytical reagent. Using site-directed mutagenesis, we have investigated in detail the role of residue H310 on pH and metal sensitivity in this luciferase. Negatively charged residues at position 310 increase the pH sensitivity and metal sensitivity; H310G considerably increases the size of the cavity, severely impacting the activity, H310R closes the cavity, and H310F considerably decreases both pH and metal sensitivities. However, no substitution completely abolished pH and metal sensitivities. The results indicate that the presence of negatively charged and basic side chains at position 310 is important for pH sensitivity and metals coordination, but not essential, indicating that the remaining side chains of E311 and E354 may still coordinate some metals in this site. Furthermore, a metal binding site search predicted that H310 mutations decrease the affinity mainly for Zn, Ni and Hg but less for Cd, and revealed the possible existence of additional binding sites for Zn, Ni and Hg.

Mutant firefly luciferase enzymes resistant to the inhibition by sodium chloride.

OBJECTIVES: Firefly luciferase, one of the most extensively studied enzymes, has numerous applications. However, luciferase activity is inhibited by sodium chloride. This study was aimed at obtaining mutant luciferase enzymes resistant to the sodium chloride inhibition. RESULTS: We first obtained two mutant luciferase enzymes whose inhibition were alleviated and determined the mutations to be Val288Ile and Glu488Val. Under medical dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level. In contrast, the single mutants, Val288Ile and Glu488Val, retained 67% and 79% of their original activity, respectively. Next, we introduced Val288Ile and Glu488Val mutations into wild-type luciferase to create a double mutant using site-directed mutagenesis. Notably, the double mutant retained its activity more than 95% of that in the absence of sodium chloride. CONCLUSIONS: The mutant luciferase, named luciferase CR, was found to retain its activity in various concentrations of sodium chloride. The luciferase CR may be extensively useful in any bioassay which includes firefly luciferase and is employed in the presence of sodium chloride.

Luciferase isozymes from the Brazilian Aspisoma lineatum (Lampyridae) firefly: origin of efficient pH-sensitive lantern luciferases from fat body pH-insensitive ancestors.

Firefly luciferases usually emit green-yellow bioluminescence at physiological pH values. However, under acidic conditions, in the presence of heavy metals and, at high temperatures they emit red bioluminescence. To understand the structural origin of bioluminescence colors and pH-sensitivity, about 20 firefly luciferases have been cloned, sequenced and investigated. The proton and metal-binding site responsible for pH- and metal sensitivity in firefly luciferases was shown to involve the residues H310, E311 and E354 in firefly luciferases. However, it is still unclear how and why pH-sensitivity arose and evolved in firefly luciferases. Here, we cloned and characterized two novel luciferase cDNAs from the fat body and lanterns of the Brazilian firefly Aspisoma lineatum. The larval fat body isozyme (AL2) has 545 residues, and displays very slow luminescence kinetics and a pH-insensitive spectrum. The adult lantern isozyme (AL1) has 548 residues, displays flash-like kinetics and pH and metal sensitive bioluminescence spectra, and is at least 10 times catalytically more efficient than AL2. Thermostability and CD studies showed that AL2 is much more stable and rigid than the AL1 isozyme. Multialignment and modelling studies show that the E310Q substitution (E310 in AL2 and Q310 in AL1) may have been critical for the origin of pH-sensitivity in firefly luciferases. The results indicate that the lantern efficient flash-emitting pH-sensitive luciferases arose from less efficient glow-type pH-insensitive luciferases found in the fat body of ancestral larval fireflies by enzyme structure flexibilization and substitution at position 310.

Characterisation of utrophin modulator SMT C1100 as a non-competitive inhibitor of firefly luciferase.

Firefly luciferase (FLuc) is a powerful tool for molecular and cellular biology, and popular in high-throughput screening and drug discovery. However, FLuc assays have been plagued with positive and negative artefacts due to stabilisation and inhibition by small molecules from a range of chemical classes. Here we disclose Phase II clinical compound SMT C1100 for the treatment of Duchenne muscular dystrophy as an FLuc inhibitor (KD of 0.40 ±â€¯0.15 µM). Enzyme kinetic studies using SMT C1100 and other non-competitive inhibitors including resveratrol and NFκBAI4 identified previously undescribed modes of inhibition with respect to FLuc's luciferyl adenylate intermediate. Employing a photoaffinity strategy to identify SMT C1100's binding site, a photolabelled SMT C1100 probe instead underwent FLuc-dependent photooxidation. Our findings support novel binding sites on FLuc for non-competitive inhibitors.

Quantum-mechanical hydration plays critical role in the stability of firefly oxyluciferin isomers: State-of-the-art calculations of the excited states.

Stabilizing mechanisms of three possible isomers (phenolate-keto, phenolate-enol, and phenol-enolate) of the oxyluciferin anion hydrated with quantum explicit water molecules in the first singlet excited state were investigated using first-principles Born-Oppenheimer molecular dynamics simulations for up to 1.8 ns (or 3.7 × 106 MD steps), revealing that the surrounding water molecules were distributed to form clear single-layered structures for phenolate-keto and multi-layered structures for phenolate-enol and phenol-enolate isomers. The isomers employed different stabilizing mechanisms compared to the ground state. Only the phenolate-keto isomer became attracted to the water molecules in its excited state and was stabilized by increasing the number of hydrogen bonds with nearby water molecules. The most stable isomer in the excited state was the phenolate-keto, and the phenolate-enol and phenol-enolate isomers were higher in energy by ∼0.38 eV and 0.57 eV, respectively, than the phenolate-keto. This was in contrast to the case of ground state in which the phenolate-enol was the most stable isomer.

Non-Steady State Analysis of Enzyme Kinetics in Real Time Elucidates Substrate Association and Dissociation Rates: Demonstration with Analysis of Firefly Luciferase Mutants.

Firefly luciferase has been widely used in biotechnology and biophotonics due to photon emission during enzymatic activity. In the past, the effect of amino acid substitutions (mutants) on the enzymatic activity of firefly luciferase has been characterized by the Michaelis constant, KM. The KM is obtained by plotting the maximum relative luminescence units (RLU) detected for several concentrations of the substrate (luciferin or luciferyl-adenylate). The maximum RLU is used because the assay begins to violate the quasi-steady state approximation when RLU decays as a function of time. However, mutations also affect the time to reach and decay from the maximum RLU. These effects are not captured when calculating the KM. To understand changes in the RLU kinetics of firefly luciferase mutants, we used a Michaelis-Menten model with the non-steady state approximation. In this model, we do not assume that the amount of enzyme-substrate complex is at equilibrium throughout the course of the experiment. We found that one of the two mutants analyzed in this study decreases not only the dissociation rate ( koff) but also the association rate ( kon) of luciferyl-adenylate, suggesting the narrowing of the structural pocket containing the catalytic amino acids. Furthermore, comparative analysis of the nearly complete oxidation of luciferyl-adenylate with wild-type and mutant firefly luciferase reveals that the total amount of photons emitted with the mutant is 50-fold larger than that with the wild type, on average. These two results together indicate that the slow supply of luciferyl-adenylate to the enzyme increases the total number of photons emitted from the substrate, luciferyl-adenylate. Analysis with the non-steady state approximation model is generally applicable when enzymatic production kinetics are monitored in real time.

Mutagenesis and Structural Studies Reveal the Basis for the Activity and Stability Properties That Distinguish the Photinus Luciferases scintillans and pyralis.

The dazzling yellow-green light emission of the common North American firefly Photinus pyralis and other bioluminescent organisms has provided a wide variety of prominent research applications like reporter gene assays and in vivo imaging methods. While the P. pyralis enzyme has been extensively studied, only recently has a second Photinus luciferase been cloned from the species scintillans. Even though the enzymes share very high sequence identity (89.8%), the color of the light they emit, their specific activity and their stability to heat, pH, and chemical denaturation are quite different with the scintillans luciferase being generally more resistant. Through the construction and evaluation of the properties of chimeric domain swapped, single point, and various combined variants, we have determined that only six amino acid changes are necessary to confer all of the properties of the scintillans enzyme to wild-type P. pyralis luciferase. Altered stability properties were attributed to four of the amino acid changes (T214N/S276T/H332N/E354N), and single mutations each predominantly changed emission color (Y255F) and specific activity (A222C). Results of a crystallographic study of the P. pyralis enzyme containing the six changes (Pps6) provide some insight into the structural basis for some of the documented property differences.

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.

A highly efficient, thermostable and cadmium selective firefly luciferase suitable for ratiometric metal and pH biosensing and for sensitive ATP assays.

Firefly luciferases have been widely used for bioanalytical purposes during the last 5 decades. They usually emit yellow-green bioluminescence and are pH-sensitive, displaying a color change to red at acidic pH and higher temperature and in the presence of heavy metals. Besides the usual applications as bioanalytical reagents and as reporter genes, firefly luciferases' pH- and metal-sensitivities have been recently harnessed for intracellular metal and pH biosensing. Previously we cloned the luciferase of the Brazilian Amydetes vivianii firefly which displays the most blue-shifted color among known firefly luciferases. Here we purified it, characterized and investigated the kinetic properties and the pH, metal and thermal sensitivities of this firefly luciferase. This luciferase displays the lowest reported KM for ATP, the highest catalytic efficiencies, and the highest thermostability among the studied recombinant beetle luciferases, making this enzyme and its cDNA an ideal reagent for sensitive ATP assays and reporter gene. The blue-shifted spectrum, higher thermostability, lower pH- and thermal-sensitivities and protein fluorescence studies indicate a more rigid active site during light emission. This enzyme displays an unmatched selective spectral sensitivity for cadmium and mercury, making it a promising ratiometric indicator of such toxic metals. Finally, the weaker thermal-sensitivity compared to other firefly luciferases makes this enzyme a better ratiometric pH indicator at temperatures above 30 °C, suitable for mammalian cell assays.

Statistical Coupling Analysis-Guided Library Design for the Discovery of Mutant Luciferases.

Directed evolution has proven to be an invaluable tool for protein engineering; however, there is still a need for developing new approaches to continue to improve the efficiency and efficacy of these methods. Here, we demonstrate a new method for library design that applies a previously developed bioinformatic method, Statistical Coupling Analysis (SCA). SCA uses homologous enzymes to identify amino acid positions that are mutable and functionally important and engage in synergistic interactions between amino acids. We use SCA to guide a library of the protein luciferase and demonstrate that, in a single round of selection, we can identify luciferase mutants with several valuable properties. Specifically, we identify luciferase mutants that possess both red-shifted emission spectra and improved stability relative to those of the wild-type enzyme. We also identify luciferase mutants that possess a >50-fold change in specificity for modified luciferins. To understand the mutational origin of these improved mutants, we demonstrate the role of mutations at N229, S239, and G246 in altered function. These studies show that SCA can be used to guide library design and rapidly identify synergistic amino acid mutations from a small library.

Pyridone Luciferins and Mutant Luciferases for Bioluminescence Imaging.

New applications for bioluminescence imaging require an expanded set of luciferase enzymes and luciferin substrates. Here, we report two novel luciferins for use in vitro and in cells. These molecules comprise regioisomeric pyridone cores that can be accessed from a common synthetic route. The analogues exhibited unique emission spectra with firefly luciferase, although photon intensities remained weak. Enhanced light outputs were achieved by using mutant luciferase enzymes. One of the luciferin-luciferase pairs produced light on par with native probes in live cells. The pyridone analogues and complementary luciferases add to a growing set of designer probes for bioluminescence imaging.

ΔFlucs: Brighter Photinus pyralis firefly luciferases identified by surveying consecutive single amino acid deletion mutations in a thermostable variant.

The bright bioluminescence catalyzed by Photinus pyralis firefly luciferase (Fluc) enables a vast array of life science research such as bio imaging in live animals and sensitive in vitro diagnostics. The effectiveness of such applications is improved using engineered enzymes that to date have been constructed using amino acid substitutions. We describe ΔFlucs: consecutive single amino acid deletion mutants within six loop structures of the bright and thermostable ×11 Fluc. Deletion mutations are a promising avenue to explore new sequence and functional space and isolate novel mutant phenotypes. However, this method is often overlooked and to date there have been no surveys of the effects of consecutive single amino acid deletions in Fluc. We constructed a large semi-rational ΔFluc library and isolated significantly brighter enzymes after finding ×11 Fluc activity was largely tolerant to deletions. Targeting an "omega-loop" motif (T352-G360) significantly enhanced activity, altered kinetics, reduced Km for D-luciferin, altered emission colors, and altered substrate specificity for redshifted analog DL-infraluciferin. Experimental and in silico analyses suggested remodeling of the Ω-loop impacts on active site hydrophobicity to increase light yields. This work demonstrates the further potential of deletion mutations, which can generate useful Fluc mutants and broaden the palette of the biomedical and biotechnological bioluminescence enzyme toolbox.

Color-shifting mutations in the C-domain of L. mingrelica firefly luciferase provide new information about the domain alternation mechanism.

We identified three color-shifting mutations-Phe467Ser, Glu490Val, and Glu490Lys-in the C-domain of the wild-type recombinant L. mingrelica luciferase. These mutations had moderate effect on the specific activity and thermal stability of the enzyme but changed the pH-dependence of its bioluminescence spectra. We constructed the model structures of the enzyme in three known conformations (open, adenylation, and oxidation conformation). The structural analysis and experimental data provided no evidences that these residues participate in structure-forming interactions in the open or oxidation conformation or that their mutations alter the overall structure of the enzyme. Given that the bioluminescence spectra reflect the microenvironment of the emitter (oxyluciferin in an electronically excited state), we concluded that the mutated residues affect the active site during the emission of light via short-range interactions. We found that it is only in the adenylation conformation that the residues Phe467 and Glu490 approach the N-domain, whereas the domain rotation associated with the oxidation conformation completely removes them from the active site. Therefore, the emission most likely occurs from the adenylation conformation.

Multiplex detection of protein-protein interactions using a next generation luciferase reporter.

Cell-based assays of protein-protein interactions (PPIs) using split reporter proteins can be used to identify PPI agonists and antagonists. Generally, such assays measure one PPI at a time, and thus counterscreens for on-target activity must be run in parallel or at a subsequent stage; this increases both the cost and time during screening. Split luciferase systems offer advantages over those that use split fluorescent proteins (FPs). This is since split luciferase offers a greater signal:noise ratio and, unlike split FPs, the PPI can be reversed upon small molecule treatment. While multiplexed PPI assays using luciferase have been reported, they suffer from low signal:noise and require fairly complex spectral deconvolution during analysis. Furthermore, the luciferase enzymes used are large, which limits the range of PPIs that can be interrogated due to steric hindrance from the split luciferase fragments. Here, we report a multiplexed PPI assay based on split luciferases from Photinus pyralis (firefly luciferase, FLUC) and the deep-sea shrimp, Oplophorus gracilirostris (NanoLuc, NLUC). Specifically, we show that the binding of the p53 tumor suppressor to its two major negative regulators, MDM2 and MDM4, can be simultaneously measured within the same sample, without the requirement for complex filters or deconvolution. We provide chemical and genetic validation of this system using MDM2-targeted small molecules and mutagenesis, respectively. Combined with the superior signal:noise and smaller size of split NanoLuc, this multiplexed PPI assay format can be exploited to study the induction or disruption of pairwise interactions that are prominent in many cell signaling pathways.

Brominated Luciferins Are Versatile Bioluminescent Probes.

We report a set of brominated luciferins for bioluminescence imaging. These regioisomeric scaffolds were accessed by using a common synthetic route. All analogues produced light with firefly luciferase, although varying levels of emission were observed. Differences in photon output were analyzed by computation and photophysical measurements. The brightest brominated luciferin was further evaluated in cell and animal models. At low doses, the analogue outperformed the native substrate in cells. The remaining luciferins, although weak emitters with firefly luciferase, were inherently capable of light production and thus potential substrates for orthogonal mutant enzymes.

Spontaneous luminescence color change in the firefly luciferase assay system.

The temporal effects of luciferase reaction luminescence have only been discussed in the context of light intensity (flash vs. glow). However, alterations in the color of the light emitted over the course of the luciferase reaction have not been reported. Here, we show a temporal change in the light color emitted during the reaction catalyzed by unmodified firefly luciferase when concentrations of one of the substrates, adenosine triphosphate (ATP), are gradually increased. The temporal color change from green to red occurs within the first few minutes of the luciferase reaction when an ATP-containing solution is either added or synthesized in situ with the aid of an autocatalytic reaction occurring simultaneously. This color change is not accompanied by pH changes. An analysis of the red and green channels demonstrates dissimilar kinetics, suggesting the co-existence of two or more temporally shifted luminescence pathways. The implications of these findings might improve dual-color biosensing/imaging protocols and influence the engineering of biophotonic systems.

Biochemical characteristics and gene expression profiles of two paralogous luciferases from the Japanese firefly Pyrocoelia atripennis (Coleoptera, Lampyridae, Lampyrinae): insight into the evolution of firefly luciferase genes.

Two paralogous genes of firefly luciferase, Luc1 and Luc2, have been isolated from the species in two subfamilies, Luciolinae and Photurinae, of the family Lampyridae. The gene expression profiles have previously been examined only in the species of Luciolinae. Here we isolated Luc1 and Luc2 genes from the Japanese firefly Pyrocoelia atripennis. This is the first report of the presence of both Luc1 and Luc2 genes in the species of the subfamily Lampyrinae and of the exon-intron structure of Luc2 in the family Lampyridae. The luminescence of both gene products peaked at 547 nm under neutral buffer conditions, and the spectrum of Luc1, but not Luc2, was red-shifted under acidic conditions, as observed for Luc2 in the Luciolinae species. The semi-quantitative reverse transcription-polymerase chain reaction suggested that Luc1 was expressed in lanterns of all the stages except eggs, while Luc2 was expressed in the non-lantern bodies of eggs, prepupae, pupae, and female adults. These expression profiles are consistent with those in the Luciolinae species. Considering the distant phylogenetic relationship between Lampyrinae and Luciolinae in Lampyridae, we propose that fireflies generally possess two different luciferase genes and the biochemical properties and gene expression profiles for each paralog are conserved among lampyrid species.

Identification and characterization of the Luc2-type luciferase in the Japanese firefly, Luciola parvula, involved in a dim luminescence in immobile stages.

Nocturnal Japanese fireflies, Luciola parvula, emit from their lanterns a yellow light, one of the most red-shifted colors found among fireflies. Previously, we isolated and characterized two different types of luciferase gene, Luc1 and Luc2, from the fireflies Luciola cruciata and Luciola lateralis; Luc1 is responsible for the green-yellow luminescence of larval and adult lanterns, whereas Luc2 is responsible for the dim greenish glow of eggs and pupal bodies. The biological role of firefly lanterns in adults is related to sexual communication, but why the eggs and pupae glow remains uncertain. In this study, we isolated the gene Luc2 from L. parvula, and compared its expression profiles and enzymatic characteristics with those of Luc1. A semi-quantitative reverse transcription polymerase chain reaction showed that Luc1 was predominantly expressed in larvae, prepupae, pupae and adults, whereas Luc2 was expressed in eggs, prepupae, pupae and adult females. Enzymatic analyses showed that the luminescent color of Luc1 matches the visual sensitivity of L. parvula eyes, whereas that of Luc2 is very different from it. These results suggest that the biological role of Luc2 expressed in immobile stages is not intraspecific communication.

Sulfoluciferin is Biosynthesized by a Specialized Luciferin Sulfotransferase in Fireflies.

Firefly luciferin is a specialized metabolite restricted to fireflies (family Lampyridae) and other select families of beetles (order Coleoptera). Firefly luciferin undergoes luciferase-catalyzed oxidation to produce light, thereby enabling the luminous mating signals essential for reproductive success in most bioluminescent beetles. Although firefly luciferin and luciferase have become widely used biotechnological tools, questions remain regarding the physiology and biochemistry of firefly bioluminescence. Here we report sulfoluciferin to be an in vivo derivative of firefly luciferin in fireflies and report the cloning of luciferin sulfotransferase (LST) from the North American firefly Photinus pyralis. LST catalyzes the production of sulfoluciferin from firefly luciferin and the sulfo-donor PAPS. Sulfoluciferin is abundant in several surveyed firefly genera as well as in the bioluminescent elaterid beetle Pyrophorus luminosus at a low level. We propose that sulfoluciferin could serve as a luciferin storage molecule in fireflies and that LST may find use as a new tool to modulate existing biotechnological applications of the firefly bioluminescent system.