Site-directed mutagenesis of firefly luciferase active site amino acids: a proposed model for bioluminescence color.

Under physiological conditions firefly luciferase catalyzes the highly efficient emission of yellow-green light from the substrates luciferin, Mg-ATP, and oxygen. In nature, bioluminescence emission by beetle luciferases is observed in colors ranging from green (approximately 530 nm) to red (approximately 635 nm), yet all known luciferases use the same luciferin substrate. In an earlier report [Branchini, B. R., Magyar, R. M., Murtiashaw, M. H., Anderson, S. M., and Zimmer, M. (1998) Biochemistry 37, 15311-15319], we described the effects of mutations at His245 on luciferase activity. In the context of molecular modeling results, we proposed that His245 is located at the luciferase active site. We noted too that the H245 mutants displayed red-shifted bioluminescent emission spectra. We report here the construction and purification of additional His245 mutants, as well as mutants at residues Lys529 and Thr343, all of which are stringently conserved in the beetle luciferase sequences. Analysis of specific activity and steady-state kinetic constants suggested that these residues are involved in luciferase catalysis and the productive binding of substrates. Bioluminescence emission spectroscopy studies indicated that point mutations at His245 and Thr343 produced luciferases that emitted light over the color range from green to red. The results of mutational and biochemical studies with luciferase reported here have enabled us to propose speculative mechanisms for color determination in firefly bioluminescence. An essential role for Thr343, the participation of His245 and Arg218, and the involvement of bound AMP are indicated.     

The role of active site residue arginine 218 in firefly luciferase bioluminescence

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate firefly luciferin by a sequence of reactions that require Mg-ATP and molecular oxygen. We had previously developed a working model of the luciferase active site based on the X-ray structure of the enzyme without bound substrates. In our model, the side chain guanidinium group of Arg218 appears to be located in close proximity to the substrate's hydroxyl group at the bottom of the luciferin binding pocket. A similar role for Arg337 also has been proposed. We report here the construction, purification, and characterization of mutant luciferases R218A, R218Q, R218K, R337Q, and R337K. Alteration of the Arg218 side chain produced enzymes with 15-20-fold increases in the Km values for luciferin. The contrasting near-normal Km values for luciferin determined with the Arg337 enzymes support our proposal that Arg218 (and not Arg337) is an essential luciferin binding site residue. Bioluminescence emission studies indicated that in the absence of a positively charged group at position 218, red bioluminescence was produced. Based on this result and those of additional fluorescence experiments, we speculate that Arg218 maintains the polarity and rigidity of the emitter binding site necessary for the normal yellow-green emission of P. pyralis luciferase. The findings reported here are interpreted in the context of the firefly luciferase X-ray structures and computational-based models of the active site.     

An alternative mechanism of bioluminescence color determination in firefly luciferase

Beetle luciferases (including those of the firefly) use the same luciferin substrate to naturally display light ranging in color from green (lambda(max) approximately 530 nm) to red (lambda(max) approximately 635 nm). In a recent communication, we reported (Branchini, B. R., Murtiashaw, M. H., Magyar, R. A., Portier, N. C., Ruggiero, M. C., and Stroh, J. G. (2002) J. Am. Chem. Soc. 124, 2112-2113) that the synthetic adenylate of firefly luciferin analogue D-5,5-dimethylluciferin was transformed into the emitter 5,5-dimethyloxyluciferin in bioluminescence reactions catalyzed by luciferases from Photinus pyralis and the click beetle Pyrophorus plagiophthalamus. 5,5-Dimethyloxyluciferin is constrained to exist in the keto form and fluoresces mainly 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, were taken as experimental support for mechanisms of firefly bioluminescence color that require only a single keto form of oxyluciferin. We report here the results of mutagenesis studies designed to determine the basis of the observed differences in bioluminescence color with the analogue adenylate. Mutants of P. pyralis luciferase putative active site residues Gly246 and Phe250, as well as corresponding click beetle residues Ala243 and Ser247 were constructed and characterized using bioluminescence emission spectroscopy and steady state kinetics with adenylate substrates. Based on an analysis of these and recently reported (Branchini, B. R., Southworth, T. L., Murtiashaw, M. H., Boije, H., and Fleet, S. E. (2003) Biochemistry 42, 10429-10436) data, we have developed an alternative mechanism of bioluminescence color. The basis of the mechanism is that luciferase modulates emission color by controlling the resonance-based charge delocalization of the anionic keto form of the oxyluciferin excited state.     

Mutagenesis evidence that the partial reactions of firefly bioluminescence are catalyzed by different conformations of the luciferase C-terminal domain

Firefly luciferase catalyzes two sequential partial reactions resulting in the emission of light. The enzyme first catalyzes the adenylation of substrate luciferin with Mg-ATP followed by the multistep oxidation of the adenylate to form the light emitter oxyluciferin in an electronically excited state. The beetle luciferases are members of a large superfamily, mainly comprised of nonbioluminescent enzymes that activate carboxylic acid substrates to form acyl-adenylate intermediates. Recently, the crystal structure of a member of this adenylate-forming family, acetyl-coenzyme A (CoA) synthetase, was determined in complex with an unreactive analogue of its acyl-adenylate and CoA [Gulick, A. M., Starai, V. J., Horswill, A. R., Homick, K. M., and Escalante-Semerena, J. C. (2003) Biochemistry 42, 2866-2873]. This structure presented a new conformation for this enzyme family, in which a significant rotation of the C-terminal domain brings residues of a conserved beta-hairpin motif to interact with the active site. We have undertaken a mutagenesis approach to study the roles of key residues of the equivalent beta-hairpin motif in Photinus pyralis luciferase (442IleLysTyrLysGlyTyrGlnVal449) in the overall production of light and the individual adenylation and oxidation partial reactions. Our results strongly suggest that Lys443 is critical for efficient catalysis of the oxidative half-reaction. Additionally, we provide evidence that Lys443 and Lys529, located on opposite sides of the C-terminal domain and conserved in all firefly luciferases, are each essential for only one of the partial reactions of firefly bioluminescence, supporting the proposal that the superfamily enzymes may adopt two different conformations to catalyze the two half-reactions.     

Biosynthesis-inspired deracemizative production of d-luciferin by combining luciferase and thioesterase

Due to the strict enantioselectivity of firefly luciferase, only d-luciferin can be used as a substrate for bioluminescence reactions. Unfortunately, luciferin racemizes easily and accumulation of nonluminous l-luciferin has negative influences on the light emitting reaction. Thus, maintaining the enantiopurity of luciferin in the reaction mixture is one of the most important demands in bioluminescence applications using firefly luciferase. In fireflies, however, l-luciferin is the biosynthetic precursor of d-luciferin, which is produced from the L-form undergoing deracemization. This deracemization consists of three successive reactions: l-enantioselective thioesterification by luciferase, in situ epimerization, and hydrolysis by thioesterase. In this work, we introduce a deracemizative luminescence system inspired by the biosynthetic pathway of d-luciferin using a combination of firefly luciferase from Luciola cruciata (LUC-G) and fatty acyl-CoA thioesterase II from Escherichia coli (TESB). The enzymatic reaction property analysis indicated the importance of the concentration balance between LUC-G and TESB for efficient d-luciferin production and light emission. Using this deracemizative luminescence system, a highly sensitive quantitative analysis method for l-cysteine was constructed. This LUC-G-TESB combination system can improve bioanalysis applications using the firefly bioluminescence reaction by efficient deracemization of D-luciferin.     

The influence of insertion of a critical residue (Arg356) in structure and bioluminescence spectra of firefly luciferase

The firefly bioluminescence reaction, which uses luciferin, Mg-ATP, and molecular oxygen to yield an electronically excited oxyluciferin, is carried out by luciferase and visible light is emitted. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Among different beetle luciferases, those from Phrixothrix railroad worm emit either yellow or red bioluminescence colors. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional Arg residue at 353, which is absent in firefly luciferases. We report here the construction and purification of a mutant at residue Arg(356), which is not conserved in beetle luciferases. By insertion of an additional residue (Arg(356)) using site-specific insertion mutagenesis in a green-emitter luciferase (Lampyris turkestanicus) the color of emitted light was changed to red and the optimum temperature of activity was also increased. Insertion of this Arg in an important flexible loop showed changes of the bioluminescence color and the luciferase reaction took place with relatively retention of its basic kinetic properties such as Km and relative activity. Comparison of native and mutant luciferases using homology modeling reveals a significant conformational change of the flexible loop in the red mutant. Movement of flexible loop brought about a new ionic interaction concomitant with a change in polarity of the emitter site, thereby leading to red emission. It is worthwhile to note that the increased optimum temperature and emission of red light might make mutant luciferase a suitable reporter for the study of gene expression and bioluminescence imaging.     

Synthesis of an N-acyl sulfamate analog of luciferyl-AMP: a stable and potent inhibitor of firefly luciferase

In the first of two half-reactions resulting in the emission of visible light, firefly luciferase forms luciferyl-adenylate from its natural substrates beetle luciferin and Mg-ATP. The acyl-adenylate is subsequently oxidized producing the light emitter oxyluciferin in an electronically excited state. In vitro, under mild conditions of temperature and pH, the acyl-adenylate intermediate is readily hydrolyzed and susceptible to oxidation. We report here the multi-step synthesis and physical and enzymatic characterization of an N-acyl sulfamate analog of luciferyl-adenylate, 5'-O-[(N-dehydroluciferyl)-sulfamoyl]-adenosine (compound 5). This represents the first example of a stable and potent (Ki = 340 nM) reversible inhibitor of firefly luciferase activity based on the structure of the natural acyl-adenylate intermediate. Additionally, we present the results of limited proteolysis studies that demonstrate that the binding of the novel acyl-adenylate analog protects luciferase from proteolysis. The findings presented here are interpreted in the context of the hypothesis that luciferase and the other enzymes in a large superfamily of adenylate-forming proteins adopt two conformations to catalyze two different partial reactions. We anticipate that the novel N-acyl sulfamate analog will be a valuable reagent in future studies designed to elucidate the role of conformational changes in firefly luciferase catalyzed bioluminescence.     

A bifunctional luminogenic substrate for two luminescent enzymes: firefly luciferase and horseradish peroxidase.

Horseradish peroxidase (HRP) catalyzes the oxidative chemiluminescent reaction of luminol, and firefly luciferase catalyzes the oxidation of firefly D-luciferin. Here we report a novel substrate, 5-(5'-azoluciferinyl)-2,3-dihydro-1,4-phthalazinedione (ALPDO), that can trigger the activity of HRP and firefly luciferase in solution because it contains both luminol and luciferin functionalities. It is synthesized by diazotization of luminol and its subsequent azo coupling with firefly luciferin. NMR spectral data show that the C5' of benzothiazole in luciferin connects the diazophthalahydrazide. The electronic absorption and fluorescence properties of ALPDO are different from those of its precursor molecules. The chemiluminescence emission spectra of the conjugate substrate display biphotonic emission characteristic of azophthalatedianion and oxyluciferin. It has an optimum pH of 8.0 for maximum activity with respect to HRP as well as luciferase. At pH 8.0 the bifunctional substrate has 12 times the activity of luminol but has 7 times less activity than the firefly luciferin-luciferase system. The specific enhancement of light emission from the cyclic hydrazide part of ALPDO helped in the sensitive assay of HRP down to 2.0 x 10(-13) M and of ATP to 1.0 x 10(-14) mol. Addition of enhancers such as firefly luciferin and p-iodophenol (PIP) to the HRP-ALPDO-H2O2 system enhanced the light output.     

Development of near-infrared firefly luciferin analogue reacted with wild-type and mutant luciferases

Interestingly, only the D-form of firefly luciferin produces light by luciferin–luciferase (L–L) reaction. Certain firefly luciferin analogues with modified structures maintain bioluminescence (BL) activity; however, all L-form luciferin analogues show no BL activity. To this date, our group has developed luciferin analogues with moderate BL activity that produce light of various wavelengths. For in vivo bioluminescence imaging, one of the important factors for detection sensitivity is tissue permeability of the number of photons emitted by L–L reaction, and the wavelengths of light in the near-infrared (NIR) range (700–900 nm) are most appropriate for the purpose. Some NIR luciferin analogues by us had performance for in vivo experiments to make it possible to detect photons from deep target tissues in mice with high sensitivity, whereas only a few of them can produce NIR light by the L–L reactions with wild-type luciferase and/or mutant luciferase. Based on the structure–activity relationships, we designed and synthesized here a luciferin analogue with the 5-allyl-6-dimethylamino-2-naphthylethenyl moiety. This analogue exhibited NIR BL emissions with wild-type luciferase (λ_max = 705 nm) and mutant luciferase AlaLuc (λ_max = 655 nm).     

Interaction of firefly luciferase with substrates and their analogs: a study using fluorescence spectroscopy methods.

The results of the author's laboratory on the interaction of Luciola mingrelica firefly luciferase with substrates and their analogs using both steady-state and time resolved fluorescence are reviewed. The contribution of fluorescence of Trp and Tyr residues of the protein to its intrinsic fluorescence spectrum was estimated. Studies of quenching of Trp and Tyr fluorescence by luciferin and ATP allowed one to determine binding constants of the luciferase with substrates and to show that the binding of one substrate to the luciferase decreases the affinity of the enzyme for the other one. Fluorescence of oxyluciferin and its analogs (dimethyl- and monomethyloxyluciferins) was shown to be a good model of native firefly bioluminescence. A comparison of the fluorescence spectra of oxyluciferin and its analogs in aqueous solutions and in the presence of the luciferase revealed specific and nonspecific effects of the microenvironment on the equilibrium between different ionic forms of oxyluciferin. An approach based on photo-physical concepts of the correlation between luminescence spectra and structure of the emitter and its microenvironment was proposed and this approach was used to analyze bioluminescence spectra of wild-type and mutant luciferases.     

Synthesis and bioluminescence of difluoroluciferin

A new synthesis route to firefly luciferin analogs was developed via the synthesis of 5′,7′-difluoroluciferin. As a luciferase substrate, it produces maximal bioluminescence at a much lower pH than is optimal for native luciferin, and at lower pH it gives much more of the red-shifted emission that is characteristic of the phenolate. These features are attributed to the enhanced acidity of the o,o-difluorophenol.     

Earthworm bioluminescence: characterization of high specific activity Diplocardia longa luciferase and the reaction it catalyzes

Diplocardia longa luciferase purified by an improved procedure differs from that first described by Bellisario et al. [Bellisario, R., Spencer, T. E., & Cormier, M. J. (1972) Biochemistry 11, 2256-2266] in having much higher specific activity (40X) and firmly bound, EPR-silent copper. Improved assay conditions suggest that this protein acts as a catalyst in a bioluminescent reaction involving the degradation of 3-(isovalerylamino)-1-hydroxypropane hydroperoxide. This substrate is formed spontaneously on the addition of hydrogen peroxide to D. longa luciferin (3-(isovalerylamino)propanal). The quantum yield of the bioluminescence for this substrate is 3%. Detailed physical and chemical analyses of high specific activity D. longa luciferase indicate that it is a large (300000 daltons), asymmetric (f/fo=1.63, with 0.4 g/g hydration), multisubunit enzyme. It contains carbohydrate (6%), lipid (2%), and copper (up to 4 mol/30000 daltons). The amino acid composition is unusual with 11% by weight of the residues being either proline or hydroxyproline.     

Human plasma gelsolin binds adenosine triphosphate

Binding studies of human plasma gelsolin with ATP were done by equilibrium dialysis. Analysis of the binding data showed that plasma gelsolin had one class of ATP binding site with Kd = 2.8 x 10(-7) M, which saturated at an ATP/gelsolin ratio of 0.6. The bioluminescent assay for ATP with luciferin and firefly luciferase confirmed that the protein contained a nucleotide as ATP.     

New Analysis Method for Protein Phosphatase Type 2A Inhibitors Using the Firefly Bioluminescence System

A new analysis method for protein phosphatase type 2A inhibitors was established that uses the firefly bioluminescence system for detection. Thus, firefly luciferin phosphate was used as a substrate, and the liberated free luciferin was determined from the amount of light emitted from the immobilized luciferase. This method was successfully used to determine the activities of known inhibitors, i.e., okadaic acid, calyculin A, microcystin-LR and tautomycin using less than 10 pmol of a sample.     

Synergistic mutations produce blue-shifted bioluminescence in firefly luciferase

Light emission from the North American firefly Photinus pyralis, which emits yellow-green (557 nm) light, is widely believed to be the most efficient bioluminescence system known, making this luciferase an excellent tool for monitoring gene expression. In a previous study designed to produce luciferases for simultaneously monitoring two gene expression events, we identified a very promising blue-shifted emitter (548 nm) that contained the mutations Val241Ile, Gly246Ala, and Phe250Ser [Branchini, B. R., Southworth, T. L., Khattak, N. F., Michelini, E., and Roda, A. (2005) Red- and green-emitting firefly luciferase mutants for bioluminescent reporter applications, Anal. Biochem. 345, 140-148]. To establish the basis of the unusual blue-shifted emission, we determined that a simple additive effect of the three individual mutations did not account for the spectral properties of the triple mutant. Instead, the bioluminescence emission spectra of two double mutants containing Phe250Ser and either Val241Ile or Gly246Ala very closely resembled that of the triple mutant. Additional mutagenesis results confirmed that the blue-shifted emission of the double mutants was determined by the synergistic behavior of active site residues. Molecular modeling studies of the Gly246Ala and Phe250Ser double mutant supported the notion that the blue-shifted emission was due to localized changes that increased the hydrophobicity at the emitter site as a result of the addition of a single methyl group at position 246. Moreover, the modeling data suggested that the Ala246 side chain remained close to the emitter through an additional H-bond between Ala246 and the hydroxyl group of Phe250, providing a possible structural basis for the synergistic behavior.     

EVOLUTION OF BIOLUMINESCENCE IN MARINE DINOFLAGELLATES

Bioluminescence is broadly distributed in marine dinoflagellates and has been intensively studied in Lingulodinium (Gonyaulax) polyedra. In this species, bioluminescence is regulated in a circadian fashion; the enzyme (luciferase) and the luciferin (substrate)‐binding protein are synthesized and degraded on a daily basis. Synthesis of both proteins is regulated at the level of translation. The L. polyedra luciferase gene is composed of three contiguous domains that are greater than 75% identical at the nucleic acid level. Possible explanations for the high degree of sequence conservation include: (1) the domains evolved through a recent duplication event; (2) the sequence similarity is maintained by a molecular process such as gene conversion; or (3) there is a functional role associated with the primary nucleic acid sequence, such as in the translational regulation of luciferase expression. The phylogenetic relationship of dinoflagellates predicted from 18S rDNA genes provides a framework for examining the molecular evolution of the regulation of luciferase expression and of genes encoding luciferase and the luciferin‐binding protein. In particular, we are examining the evolution of the circadian rhythm of bioluminescence and of luciferase abundance, the presence/absence of the luciferin‐binding protein, and the molecular structure of the luciferase gene. We anticipate that this approach will distinguish between regions of the luciferase molecule that are conserved for enzyme function versus those concerned with the regulation of protein expression. In addition, it will provide insight into the evolution of the regulatory processes and pathways.     

中国萤火虫云南窗萤荧光素酶cDNA的克隆表达和序列分析

从一种来自中国日行性萤火虫(云南窗萤)发光器官mRNA中克隆、测序并表达了有功能的荧光素酶.云南窗萤荧光素酶的cDNA序列有1647个碱基,编码548个氨基酸残基.从推测得到的氨基酸序列的比对分析得出:云南窗萤的荧光素酶与来自Lampyris noctiluca,L.turkestanicus和Nyctophila cf.caucasica三种萤火虫的荧光素酶有97.8%的序列一致性.从推测得出的氨基酸序列进行系统发育分析,其结果表明:云南窗萤和Lampyris Nyctophila聚在一起,与同属的发光强夜行性的萤火虫不形成的单系.云南窗萤荧光素酶在大肠杆菌中表达的条带大约70kDa,并且在有荧光素存在时发出黄绿色荧光.对荧光素酶的结构模拟和分析表明,云南窗萤荧光素酶基因的氨基端和羧基端结构域之间的裂沟处存在这5个多肽环,这正是从其他荧光素酶推测得到的催化荧光反应时的底物结合位点.云南窗萤和窗萤属的其他3种萤火虫的荧光素酶卡目比,有13个不同氨基酸位点,位于模拟分子结构的表面.对于这些多肽环、不刚氨基酸残基和晶体结构的进一步研究有利于解释日行和夜行性萤火虫荧光素酶的差异.     

Continous monitoring of ATP-converting reactions by purified firefly luciferase.

The time course of the bioluminescence obtained with a partially purified firefly luciferase preparation has been studied. At ATP levels less than 10^?6m the light emission could be maintained essentially constant for several minutes, if the luciferase was not subjected to product inhibition or other inactivating processes. This could be achieved by performing the reaction at appropriate pH and concentration of luciferin and luciferase. Under these conditions continuous measurement of light emission may be used for nondestructive monitoring of ATP-converting reactions, since the emission will be proportional to the ATP concentration in each instant. The continuous monitoring of ATP concentration by firefly luciferase was used for kinetic determination of enzymes and metabolites and for endpoint analysis of metabolites. It was found to be extremely sensitive and convenlent for routine applications.     

Mathematical Model of the Firefly Luciferase Complementation Assay Reveals a Non-Linear Relationship between the Detected Luminescence and the Affinity of the Protein Pair Being Analyzed

The firefly luciferase complementation assay is widely used as a bioluminescent reporter technology to detect protein-protein interactions in vitro, in cellulo, and in vivo. Upon the interaction of a protein pair, complemented firefly luciferase emits light through the adenylation and oxidation of its substrate, luciferin. Although it has been suggested that kinetics of light production in the firefly luciferase complementation assay is different from that in full length luciferase, the mechanism behind this is still not understood. To quantitatively understand the different kinetics and how changes in affinity of a protein pair affect the light emission in the assay, a mathematical model of the in vitro firefly luciferase complementation assay was constructed. Analysis of the model finds that the change in kinetics is caused by rapid dissociation of the protein pair, low adenylation rate of luciferin, and increased affinity of adenylated luciferin to the enzyme. The model suggests that the affinity of the protein pair has an exponential relationship with the light detected in the assay. This relationship causes the change of affinity in a protein pair to be underestimated. This study underlines the importance of understanding the molecular mechanism of the firefly luciferase complementation assay in order to analyze protein pair affinities quantitatively.     

Few substitutions affect the bioluminescence spectra of Phrixotrix (Coleoptera: Phengodidae) luciferases: a site-directed mutagenesis survey.

Phrixotrix (railroad worm) luciferases produce bioluminescence in the green and red regions of the spectrum, depending on the location of the lanterns, and are the only luciferases naturally producing red bioluminescence. Comparison of the luciferase sequences showed a set of substitutions that could be involved in bioluminescence colour determination: (a) unique substitutions in the red luciferase replacing otherwise invariant residues; (b) conserved basic residues in the green-yellow emitting luciferases; and (c) an additional R353 residue in red-emitting luciferase (Viviani et al., 1999). To investigate whether these sites have a functional role in bioluminescence colour determination, we performed a site-directed mutagenesis. Natural substitutions in the region 220-344 and residues in the putative luciferin-binding site were also investigated. With the exception of the previously identified substitution of R215 and T226 (Viviani et al., 2002), which display dramatic red-shift effects on the spectrum of green-yellow-emitting luciferases, only a few substitutions had a moderate effect on the spectrum of the green-emitting luciferase. In contrast, no single substitution affected the spectrum of the red-emitting luciferase. The results suggest that the identity of the active site residues is not so critical for determining red bioluminescence in PxRE luciferase. Rather, the conformation assumed during the emitting step could be critical to set up proper interactions with excited oxyluciferin.