Luciferin Binding Research Articles

The crucial role of Y109 and R162 as catalytic residues of nanoKAZ: insights from molecular docking, molecular dynamics simulation, and quantum chemical investigations.

nanoKAZ is a compact luciferase that exhibits intense blue light emission when it catalyzes the substrate Furimazine (FMZ) as a luciferin, making it an excellent candidate as a reporter protein. However, the specific catalytic residues and mechanism of nanoKAZ have not been revealed. Recently, the structure of nanoKAZ was determined, and it was observed that the luminescent properties changed when FMZ analogs with naphthalene replacing benzene were used. It is speculated that the substituted naphthalene may influence the interaction between the catalytic residues and luciferins, thereby affecting the energy of the emitted light signal. Therefore, the primary objective of this study is to analyze and compare the molecular recognition between nanoKAZ and FMZ along with its four activity-altered naphthalene analogs, with aiming to identify the catalytic residues. Molecular docking was employed to construct all nanoKAZ-luciferin models, followed by a 500ns molecular dynamics simulation. The simulation trajectory was subjected to MM/PBSA analysis to identify crucial residues that contribute significantly to luciferin binding. In the result, two polar residues Y109, and R162 were identified as active residues as their notable contributions to the binding energy. Subsequently, an oxygen molecule was introduced into the local region of the nanoKAZ-FMZ complex and followed with quantum chemical calculations (semiempirical and DFT methods were used) to investigate the catalysis details. The results illustrated the involvement of Y109 and R162 in the oxygenation of FMZ, leading to the formation of dioxetanone, which has been suggested as an important intermediate in the oxidation process among various luciferins sharing the same functional group as FMZ.

Label-free MS/MS analyses of the dinoflagellate Lingulodinium identifies rhythmic proteins facilitating adaptation to a diurnal LD cycle

Protein levels were assessed in the dinoflagellate Lingulodinium polyedra over the course of a diurnal cycle using a label-free LC-MS/MS approach. Roughly 1700 proteins were quantitated in a triplicate dataset over a daily period, and 13 were found to show significant rhythmic changes. Included among the proteins found to be most abundant at night were the two bioluminescence proteins, luciferase and luciferin binding protein, as well as a proliferating cell nuclear protein involved in the nightly DNA replication. Aconitase and a pyrophosphate fructose-6-phosphate-1-phosphotransferase were also found to be more abundant at night, suggestive of an increased ability to generate ATP by glucose catabolism when photosynthesis does not occur. Among the proteins more abundant during the day were found a 2-epi-5-epi-valiolone synthase, potentially involved in synthesis of mycosporin-like amino acids that can act as a “microbial sunscreen”, and an enzyme synthesizing vitamin B6 which is known to protect against oxidative stress. A lactate oxidoreductase was also found to be more abundant during the day, perhaps to counteract the pH changes due to carbon fixation by facilitating conversion of pyruvate to lactate. This unbiased proteomic approach reveals novel insights into the daily metabolic changes of this dinoflagellate. Furthermore, the observation that only a limited number of proteins vary support a model where metabolic flux through pathways can be controlled by variations in a select few, possibly rate limiting, steps. Data are available via ProteomeXchange with identifier PXD006994.

Molecular and biochemical basis for the loss of bioluminescence in the dinoflagellate Noctiluca scintillans along the west coast of the USA.

The globally distributed heterotrophic dinoflagellate Noctiluca scintillans (Macartney) Kofoid & Swezy is well known for its dense blooms and prominent displays of bioluminescence. Intriguingly, along the west coast of the USA its blooms are not bioluminescent. We investigated the basis for the regional loss of bioluminescence using molecular, cellular and biochemical analyses of isolates from different geographic regions. Prominent differences of the non-bioluminescent strains were: (1) the fused luciferase and luciferin binding protein gene (lcf/lbp) was present but its transcripts were undetectable; (2) lcf/lbp contained multiple potentially deleterious mutations; (3) the substrate luciferin was absent, based on the lack of luciferin blue autofluorescence and the absence of luciferin derived metabolites; (4) although the cells possessed scintillons, the vesicles that contain the luminescent chemistry, electron microscopy revealed additional scintillon-like vesicles with an atypical internal structure; (5) cells isolated from the California coast were 43% smaller in size than bioluminescent cells from the Gulf of Mexico. Phylogenetic analyses based on the large subunit of rDNA did not show divergence of the non-bioluminescent population in relation to other bioluminescent N. scintillans from the Pacific Ocean and Arabian Sea. Our study demonstrates that gene silencing and the lack of the luciferin substrate have resulted in the loss of a significant dinoflagellate functional trait over large spatial scales in the ocean. As the bioluminescence system of dinoflagellates is well characterized, non-bioluminescent N. scintillans is an ideal model to explore the evolutionary and ecological mechanisms that lead to intraspecific functional divergence in natural dinoflagellate populations.

Phrixotrix luciferase and 6\u2032-aminoluciferins reveal a larger luciferin phenolate binding site and provide novel far-red combinations for bioimaging purposes

How the unique luciferase of Phrixothrix hirtus (PxRE) railroad worm catalyzes the emission of red bioluminescence using the same luciferin of fireflies, remains a mystery. Although PxRE luciferase is a very attractive tool for bioanalysis and bioimaging in hemoglobin rich tissues, it displays lower quantum yield (15%) when compared to green emitting luciferases (>40%). To identify which parts of PxRE luciferin binding site (LBS) determine bioluminescence color, and to develop brighter and more red-shifted emitting luciferases, we compared the effects of site-directed mutagenesis and of larger 6′-substituted aminoluciferin analogues (6′-morpholino- and 6′-pyrrolidinyl-LH) on the bioluminescence properties of PxRE and green-yellow emitting beetle luciferases. The effects of mutations in the benzothiazolyl and thiazolyl parts of PxRE LBS on the KM and catalytic efficiencies, indicated their importance for luciferin binding and catalysis. However, the absence of effects on the bioluminescence spectrum indicated a less interactive LBS in PxRE during light emission. Mutations at the bottom of LBS of PxRE blue-shifted the spectra and increased catalytic efficiency, suggesting that lack of interactions of this part of LBS with excited oxyluciferin phenolate underlie red light emission. The much higher bioluminescence activity and red-shifted spectra of PxRE luciferase with 6′-morpholino- (634 nm) and 6′-pyrrolidinyl-luciferins (644 nm), when compared to other beetle luciferases, revealed a larger luciferin phenolate binding pocket. The size and orientation of the side-chains of L/I/H348 are critical for amino-analogues accommodation and modulate bioluminescence color, affecting the interactions and mobility of excited oxyluciferin phenolate. The PxRE luciferase and 6′-aminoluciferins provide potential far-red combinations for bioimaging applications.

Biosynthetic bifunctional enzyme complex with high-efficiency luciferin-recycling to enhance the bioluminescence imaging.

Firefly luciferase is a prominent reporter on molecular imaging with the advantage of longer wavelength on light emission and the ATP linear correlation, which makes it useful in most of current bioluminescence imaging model. However, the utility of this biomaterial was limited by the signal intensity and stability which are respectively affected by enzyme activity and substrate consumption. This study demonstrated a series of novel synthetic bifunctional enzyme complex of Firefly luciferase (Fluc) and Luciferin-regenerating enzyme (LRE). A peptide linker library was constructed for the fusion strategy on biosynthesis. The findings of both experimental data and structural simulation demonstrated that the intervention of fused LRE remarkably improve the stability of in vitro bioluminescence signal through luciferin recycling; and revealed the competitive relationship of Fluc and LRE on luciferin binding: Fluc performed higher activity with one copy number of rigid linker (EAAAK) at the C terminal while LRE acted more efficiently with two copy numbers of flexible linker (GGGGS) at the N terminal. With the advantage of signal intensity and stability, this fused bifunctional enzyme complex may expand the application of firefly luciferase to in vitro bioluminescence imaging.

MiRNAs Do Not Regulate Circadian Protein Synthesis in the Dinoflagellate Lingulodinium polyedrum.

Dinoflagellates have been shown to express miRNA by bioinformatics and RNA blot (Northern) analyses. However, it is not yet known if miRNAs are able to alter gene expression in this class of organisms. We have assessed the possibility that miRNA may mediate circadian regulation of gene expression in the dinoflagellate Lingulodinium polyedrum using the Luciferin Binding Protein (LBP) as a specific example. LBP is a good candidate for regulation by miRNA since mRNA levels are constant over the daily cycle while protein synthesis is restricted by the circadian clock to a period of several hours at the start of the night phase. The transcriptome contains a potential DICER and an ARGONAUTE, suggesting the machinery for generating miRNAs is present. Furthermore, a probe directed against an abundant Symbiodinium miRNA cross reacts on Northern blots. However, L. polyedrum has no small RNAs detectable by ethidium bromide staining, even though higher plant miRNAs run in parallel are readily observed. Illumina sequencing of small RNAs showed that the majority of reads did not have a match in the L. polyedrum transcriptome, and those that did were almost all sense strand mRNA fragments. A direct search for 18–26 nucleotide long RNAs capable of forming duplexes with a 2 base 3’ overhang detected 53 different potential miRNAs, none of which was able to target any of the known circadian regulated genes. Lastly, a microscopy-based test to assess synthesis of the naturally fluorescent LBP in single cells showed that neither double-stranded nor antisense lbp RNA introduced into cells by microparticle bombardment prior to the time of LBP synthesis were able to reduce the amount of LBP produced. Taken together, our results indicate that circadian control of protein synthesis in L. polyedrum is not mediated by miRNAs.

Characterization and Subcellular Localization of Hv1 in Lingulodinium Polyedrum Confirms its Role in Bioluminescence

In 1972, J. Woodland Hastings and colleagues predicted the existence of a proton selective channel that opens in response to depolarizing voltage (HV1) across the vacuole membrane of bioluminescent dinoflagellates and conducts protons into specialized luminescence compartments (scintillons), thus causing the pH drop that triggers the light flash. RNA-Seq data from several luminescent dinoflagellate species provided candidate HV1 genes. When expressed in mammalian cells, the predicted HV1 from Lingulodinium polyedrum displays the hallmark properties of bona fide proton channels, including time-dependent opening with depolarization, perfect proton selectivity, and characteristic ΔpH dependent gating. RT-PCR and Western blotting confirm expression of HV1 in L. polyedrum. Fluorescence confocal microscopy of L. polyedrum cells stained with antibodies to luminescence proteins luciferase and luciferin binding protein and to HV1 reveal structures consistent with HV1's proposed function in bioluminescence.[Supported by NSF: MCB-1242985, NIH: GM102336, ⋯]

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 T(69)R 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 (K(77)E/T(69)R) increased the amount of bioluminescent signal more than two-fold over a short time. In addition, G(75)E, K(77)E and G(75)E/T(69) R mutants did not improve luciferin-luciferase in vitro bioluminescence. Based on our results, addition of K(77)E/G(75)E and K(77)E/G(75)E/T(69)R 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.

Bioluminescence of beetle luciferases with 6'-amino-D-luciferin analogues reveals excited keto-oxyluciferin as the emitter and phenolate/luciferin binding site interactions modulate bioluminescence colors.

Beetle luciferases produce different bioluminescence colors from green to red using the same d-luciferin substrate. Despite many studies of the mechanisms and structural determinants of bioluminescence colors with firefly luciferases, the identity of the emitters and the specific active site interactions responsible for bioluminescence color modulation remain elusive. To address these questions, we analyzed the bioluminescence spectra with 6'-amino-D-luciferin (aminoluciferin) and its 5,5-dimethyl analogue using a set of recombinant beetle luciferases that naturally elicit different colors and different pH sensitivities (pH-sensitive, Amydetes vivianii λmax=538 nm, Macrolampis sp2 λmax=564 nm; pH-insensitive, Phrixotrix hirtus λmax=623 nm, Phrixotrix vivianii λmax=546 nm, and Pyrearinus termitilluminans λmax=534 nm), a luciferase-like enzyme (Tenebrionidae, Zophobas morio λmax=613 nm), and mutants of C311 (S314). The green-yellow-emitting luciferases display red-shifted bioluminescence spectra with aminoluciferin in relation to those with D-luciferin, whereas the red-emitting luciferases displayed blue-shifted spectra. Bioluminescence spectra with 5,5-dimethylaminoluciferin, in which enolization is blocked, were almost identical to those of aminoluciferin. Fluorescence probing using 2-(4-toluidino)naphthalene-6-sulfonate and inference with aminoluciferin confirm that the luciferin binding site of the red-shifted luciferases is more polar than in the case of the green-yellow-emitting luciferases. Altogether, the results show that the keto form of excited oxyluciferin is the emitter in beetle bioluminescence and that bioluminescence colors are essentially modulated by interactions of the 6'-hydroxy group of oxyluciferin and basic moieties under the influence of the microenvironment polarity of the active site: a strong interaction between a base moiety and oxyluciferin phenol in a hydrophobic microenvironment promotes green-yellow emission, whereas a more polar environment weakens such interaction promoting red shifts. In pH-sensitive luciferases, a pH-mediated switch from a closed hydrophobic conformation to a more open polar conformation promotes the typical red shift.

Diversity of the Luciferin Binding Protein Gene in Bioluminescent Dinoflagellates – Insights from a New Gene in Noctiluca scintillans and Sequences from Gonyaulacoid Genera

Dinoflagellate bioluminescence systems operate with or without a luciferin binding protein, representing two distinct modes of light production. However, the distribution, diversity, and evolution of the luciferin binding protein gene within bioluminescent dinoflagellates are not well known. We used PCR to detect and partially sequence this gene from the heterotrophic dinoflagellate Noctiluca scintillans and a group of ecologically important gonyaulacoid species. We report an additional luciferin binding protein gene in N. scintillans which is not attached to luciferase, further to its typical combined bioluminescence gene. This supports the hypothesis that a profound re-organization of the bioluminescence system has taken place in this organism. We also show that the luciferin binding protein gene is present in the genera Ceratocorys, Gonyaulax, and Protoceratium, and is prevalent in bioluminescent species of Alexandrium. Therefore, this gene is an integral component of the standard molecular bioluminescence machinery in dinoflagellates. Nucleotide sequences showed high within-strain variation among gene copies, revealing a highly diverse gene family comprising multiple gene types in some organisms. Phylogenetic analyses showed that, in some species, the evolution of the luciferin binding protein gene was different from the organism's general phylogenies, highlighting the complex evolutionary history of dinoflagellate bioluminescence systems.

Cold-Induced Cysts of the Photosynthetic DinoflagellateLingulodinium polyedrumHave an Arrested Circadian Bioluminescence Rhythm and Lower Levels of Protein Phosphorylation

Dinoflagellates are microscopic, eukaryotic, and primarily marine plankton. Temporary cyst formation is a well-known physiological response of dinoflagellate cells to environmental stresses. However, the molecular underpinnings of cold-induced cyst physiology have never been described. Cultures of the photosynthetic dinoflagellate Lingulodinium polyedrum readily form temporary cysts when placed at low (8°C±1°C) temperature and excyst to form normal motile cells following a return to normal temperature (18°C±1°C). The normal circadian bioluminescence rhythm and the expected changes in Luciferin Binding Protein abundance were arrested in L. polyedrum cysts. Furthermore, after excystment, the bioluminescence rhythm initiates at a time corresponding to zeitgeber 12, independent of the time when the cells encysted. Phosphoprotein staining after two-dimensional polyacrylamide gel electrophoresis, as well as column-based phosphoprotein enrichment followed by liquid chromatography tandem mass spectrometry, showed cyst proteins are hypophosphorylated when compared with those from motile cells, with the most marked decreases found for predicted Casein Kinase2 target sites. In contrast to the phosphoproteome, the cyst proteome is not markedly different from motile cells, as assessed by two-dimensional polyacrylamide gel electrophoresis. In addition to changes in the phosphoproteome, RNA sequencing revealed that cysts show a significant decrease in the levels of 132 RNAs. Of the 42 RNAs that were identified by sequence analysis, 21 correspond to plastid-encoded gene products and 11 to nuclear-encoded cell wall/plasma membrane components. Our data are consistent with a model in which the highly reduced metabolism in cysts is achieved primarily by alterations in the phosphoproteome. The stalling of the circadian rhythm suggests temporary cysts may provide an interesting model to address the circadian system of dinoflagellates.

A Route from Darkness to Light: Emergence and Evolution of Luciferase Activity in AMP-CoA-Ligases Inferred from a Mealworm Luciferase-like Enzyme

The origin of luciferases and of bioluminescence is enigmatic. In beetles, luciferases seem to have evolved from AMP-CoA-ligases. How the new oxygenase luminogenic function originated from AMP-ligases leading to luciferases is one of the most challenging mysteries of bioluminescence. Comparison of the cloned luciferase-like enzyme from the nonluminescent Zophobas morio mealworm and beetle luciferases showed that the oxygenase activity may have emerged as a stereoselective oxidative drift with d-luciferin, a substrate that cannot be easily thioesterified to CoA as in the case of the l-isomer. While the overall kcat displayed by beetle luciferases is orders of magnitude greater than that of the luciferase-like enzyme, the respective oxidation rates and quantum yields of bioluminescence are roughly similar, suggesting that the rate constant of the AMP-ligase activity exerted on the new d-luciferin substrate in beetle protoluciferases was the main enzymatic property that suffered optimization during the evolution of luciferases. The luciferase-like enzyme and luciferases boost the rate of luciferyl-adenylate chemiluminescent oxidation by factors of 10(6) and 10(7), respectively, as compared to the substrate spontaneous oxidation in buffer. A similar enhancement of luciferyl-adenylate chemiluminescence is provided by nucleophilic aprotic solvents, implying that the peptide bonds in the luciferin binding site of beetle luciferase could provide a similar catalytically favorable environment. These data suggest that the luciferase-like enzyme and other similar AMP-ligases are potential alternative oxygenases. Site-directed mutagenesis studies of the luciferase-like enzyme and the red light-producing luciferase of Phrixotrix hirtus railroadworm confirm here a critical role for T/S345 in luciferase function. Mutations such as I327T/S in the luciferase-like enzyme, which simultaneously increases luciferase activity and promotes blue shifts in the emission spectrum, could have been critical for evolving functional bioluminescence from red-emitting protoluciferases. Through the combination of I327T/S mutations and N-terminal fusion, the luminescence activity of this enzyme was increased to visible levels, with the development of a totally new orange-emitting luciferase. These results open the possibility of engineering luciferase activity in a set of AMP-CoA-ligases.

The Luciferin Binding Site Residues C/T311 (S314) Influence the Bioluminescence Color of Beetle Luciferases through Main-Chain Interaction with Oxyluciferin Phenolate

Beetle luciferases emit different bioluminescence colors from green to red; however, no clear relationship between the identity of the luciferin binding site residues and bioluminescence colors was found in different luciferases, and it is unclear whether critical interactions affecting emission spectra occur on the thiazolyl or on the benzothiazolyl sides of the luciferin binding site. Through homology modeling and site-directed mutagenesis using our multicolor set of beetle luciferases (Pyrearinus termitilluminans larval click beetle, Pte, λ(max) = 534 nm; Phrixothrix hirtus railroad worm red emitting, PxRE, λ(max) = 623 nm; and Macrolampis sp2 firefly, Mac, λ(max) = 564 nm), we show that the residues C/T311 (S314) play an important role in bioluminescence color determination. Modeling studies indicate that the main-chain carbonyls of these residues are close to both oxyluciferin phenolate and AMP, whereas the side chains pack against second-shell residues. The C311(S314)A mutation considerably red shifts the spectra of the green-yellow-emitting luciferases (Pte λ(max) = 534 to 590 nm; Mac λ(max) = 564 to 583/613 nm) and affects the K(M) values for luciferin and ATP, but not the spectrum of the red-emitting luciferase. On the other hand, whereas the exchange between C/T311 (S314) caused smaller effects on the emission spectra of green-yellow-emitting luciferases, the C311T substitution (naturally found in green-emitting railroad worm luciferases) resulted in the largest reported blue shift in P. hirtus red-emitting luciferase (λ(max) = 623 to 606 nm). Altogether, these results indicate that the stability of residues C/T311 (S314) and the size of the cavity around oxyluciferin phenolate affect bioluminescence colors and suggest, for the first time, the occurrence of a critical interaction between main-chain carbonyls of position 311 (314) residues and oxyluciferin phenolate.

Structural evolution of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase) through site-directed mutagenesis of the luciferin binding site

The structural origin and evolution of bioluminescent activity of beetle luciferases from AMP/CoA ligases remains a mystery. Previously we cloned the luciferase-like enzyme from Zophobas morio mealworm, a reasonable protoluciferase model that could shine light on this mystery. Kinetic characterization and studies with D- and L-luciferin and their adenylates showed that stereoselectivity constitutes a critical feature for the origin of luciferase activity in AMP/CoA ligases. Comparison of the primary structures and modeling studies of this protoluciferase and the three main families of beetle luciferases showed that the carboxylic acid substrate binding site of this enzyme is smaller and more hydrophobic than the luciferin binding site of beetle luciferases, showing several substitutions of otherwise conserved residues. Thus, here we performed a site-directed mutagenesis survey of the carboxylic binding site motifs of the protoluciferase by replacing their residues by the respective conserved ones found in beetle luciferases in order to identify the structural determinants of luciferase/oxygenase activity. Although most of the substitutions had negative impact on the luminescence activity of the protoluciferase, only the substitution I327T improved the luminescence activity, resulting in a broad and 15 nm blue-shifted luminescence spectrum. Such substitution indicates the importance of the loop motif 322YGMSEI327 (341YGLTETT347 in Photinus pyralis luciferase) for luciferase activity, and indicates a possible route for the evolution of bioluminescence function of beetle luciferases.

The origin of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase): luciferin stereoselectivity as a switch for the oxygenase activity

Beetle luciferases evolved from AMP/CoA-ligases. However, it is unclear how the new luciferase activity evolved. In order to clarify this question, we compared the luminescence and catalytic properties of a recently cloned luciferase-like enzyme from Zophobas mealworm, an AMP/CoA-ligase displaying weak luminescence activity, with those of cloned luciferases from the three main families of luminescent beetles: Phrixthrix hirtus railroad worm; Pyrearinus termitilluminans click beetle and Photinus pyralis firefly. The catalytic constant of the mealworm enzyme was 2-4 orders of magnitude lower than that of beetle luciferases, but 3 orders of magnitude above the non-catalyzed chemiluminescence of luciferyl-adenylate in buffer. Studies with D- and L-luciferin and their adenylates show that the luminescence reaction of the luciferase-like enzyme and beetle luciferases are stereoselective for D-luciferin and its adenylate, and that the selectivity is determined mainly at the adenylation step. Modelling studies showed that the luciferin binding site cavity of this enzyme is smaller and more hydrophobic than that of beetle luciferases. Therefore Zophobas mealworm enzyme displays true luciferase activity, keeping the attributes of an ancient protoluciferase. These results suggest that stereoselectivity for D-luciferin may have been a key event for the origin of oxygenase/luciferase activity in AMP/CoA-ligases, and that efficient luciferase activity may have further evolved mainly by increasing the catalytic constant of the oxidative reaction and the quantum yield of bioluminescence.

Pyrearinus termitilluminans larval click beetle luciferase: active site properties, structure and function relationships and comparison with other beetle luciferases

Several beetle luciferases have been cloned and sequenced. However, most studies on structure and function relationships and bioanalytical applications were done with firefly luciferases, which are pH sensitive. Several years ago we cloned Pyrearinus termitilluminans larval click beetle luciferase, which displays the most blue-shifted bioluminescence among beetle luciferases and is pH insensitive. This enzyme was expressed in E. coli, purified, and its properties investigated. This luciferase shows slower luminescence kinetics, K(M) values comparable to other beetle luciferases and high catalytic constant. Fluorescence studies with 8-anilino-1-naphtalene-sulfonic acid (1,8-ANS) and modeling studies suggest that the luciferin binding site of this luciferase is very hydrophobic, supporting the solvent and orientation polarizability effects as determining mechanisms for bioluminescence colors. Although pH insensitive in the range between pH 6-8, at pH 10 this luciferase displays a remarkable red-shift and broadening of the bioluminescence spectrum. Modeling studies suggest that the residue C312 may play an important role in bioluminescence color modulation. Compared to other beetle luciferases, Pyrearinus termitilluminans luciferase also displays higher thermostability and sustained luminescence in a bacterial cell environment, which makes this luciferase particularly suitable for in vivo cell analysis and bioimaging.

A Basis for Reduced Chemical Library Inhibition of Firefly Luciferase Obtained from Directed Evolution

We measured the "druggability" of the ATP-dependent luciferase derived from the firefly Photuris pennsylvanica that was optimized using directed evolution (Ultra-Glo, Promega). Quantitative high-throughput screening (qHTS) was used to determine IC(50)s of 198899 samples against a formulation of Ultra-Glo luciferase (Kinase-Glo). We found that only 0.1% of the Kinase-Glo inhibitors showed an IC(50) < 10 microM compared to 0.9% found from a previous qHTS against the firefly luciferase from Photinus pyralis (lucPpy). Further, the maximum affinity identified in the lucPpy qHTS was 50 nM, while for Kinase-Glo this value increased to 600 nM. Compounds with interactions stretching outside the luciferin binding pocket were largely lost with Ultra-Glo luciferase. Therefore, Ultra-Glo luciferase will show less compound interference when used as an ATP sensor compared to lucPpy. This study demonstrates the power of large-scale quantitative analysis of structure-activity relationships (>100K compounds) in addressing important questions such as a target's druggability.

N-Alkylated 6′-Aminoluciferins Are Bioluminescent Substrates for Ultra-Glo and QuantiLum Luciferase: New Potential Scaffolds for Bioluminescent Assays

A set of 6'-alkylated aminoluciferins are shown to be bioluminescent substrates for Ultra-Glo and QuantiLum luciferases. These studies demonstrate that both the engineered and wild-type firefly luciferases tolerate much greater steric bulk at the 6' position of luciferin than has been previously reported. The nature of the alkyl substituent strongly affects the strength of the bioluminescent signal, which varies widely based on size, shape, and charge. Several compounds were observed to generate more light than the corresponding unsubstituted 6'-aminoluciferin. Determination of Michaelis-Menten constants for the substrates with Ultra-Glo indicated that the variation arises primarily from differences in V max, ranging from 1.33 x 10 (4) to 332 x 10 (4) relative light units, but in some cases K m (0.73-10.8 microM) also plays a role. Molecular modeling results suggest that interactions of the side chain with a hydrogen-bonding network at the base of the luciferin binding pocket may influence substrate-enzyme binding.

Interaction of Anesthetics with the Rho GTPase Regulator Rho GDP Dissociation Inhibitor

The physiological effects of anesthetics have been ascribed to their interaction with hydrophobic sites within functionally relevant CNS proteins. Studies have shown that volatile anesthetics compete for luciferin binding to the hydrophobic substrate binding site within firefly luciferase and inhibit its activity (Franks, N. P., and Lieb, W. R. (1984) Nature 310, 599-601). To assess whether anesthetics also compete for ligand binding to a mammalian signal transduction protein, we investigated the interaction of the volatile anesthetic, halothane, with the Rho GDP dissociation inhibitor (RhoGDIalpha), which binds the geranylgeranyl moiety of GDP-bound Rho GTPases. Consistent with the existence of a discrete halothane binding site, the intrinsic tryptophan fluorescence of RhoGDIalpha was quenched by halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) in a saturable, concentration-dependent manner. Bromine quenching of tryptophan fluorescence is short-range and W192 and W194 of the RhoGDIalpha are located within the geranylgeranyl binding pocket, suggesting that halothane binds within this region. Supporting this, N-acetyl-geranylgeranyl cysteine reversed tryptophan quenching by halothane. Short chain n-alcohols ( n < 6) also reversed tryptophan quenching, suggesting that RhoGDIalpha may also bind n-alkanols. Consistent with this, E193 was photolabeled by 3-azibutanol. This residue is located in the vicinity of, but outside, the geranylgeranyl chain binding pocket, suggesting that the alcohol binding site is distinct from that occupied by halothane. Supporting this, N-acetyl-geranylgeranyl cysteine enhanced E193 photolabeling by 3-azibutanol. Overall, the results suggest that halothane binds to a site within the geranylgeranyl chain binding pocket of RhoGDIalpha, whereas alcohols bind to a distal site that interacts allosterically with this pocket.

False Positives in a Reporter Gene Assay: Identification and Synthesis of Substituted N-Pyridin-2-ylbenzamides as Competitive Inhibitors of Firefly Luciferase

Luciferase reporter-gene assays are a commonly used technique in high-throughput screening campaigns. In this study, we report on a luciferase inhibitor (1), which emerged from an antagonistic G protein-coupled receptor luciferase reporter-gene assay screen. Instead of displaying receptor activity, compound 1 was shown to potently inhibit luciferase in an in vitro enzymatic assay with an IC50 value of 1.7 +/- 0.1 microM. In addition, 1 was a competitive inhibitor with respect to the substrate luciferin. A database search yielded another inhibitor (3) with a similar N-pyridin-2-ylbenzamide core. Subsequently, several analogues were prepared to investigate the structure-activity relationships of these luciferase inhibitors. This yielded the most potent inhibitor of this series (6) with an IC50 value of 0.069 +/- 0.01 microM. Further molecular modeling studies suggested that 6 can be accommodated in the luciferin binding site. This paper is meant to alert users of luciferase reporter-gene assays for possible false positive hits including highly "druglike" molecules due to direct luciferase inhibition.