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The crystal structure of Photinus pyralis luciferase shows a unique molecular architecture consisting of a large N-terminal domain and a small C-terminal domain which is separated by a wide cleft. Protein engineering methods attempts to design the peptide linkers that make a connection between different protein domains or subunits to allow for separating domains and improve kinetics and structural features of proteins. In regard to this; introduction of a flexible linker at split point of luciferase which has a strong self-association activity, may leads to conformational change and improve general flexibility of protein. In this study, two flexible linkers in the split point of luciferase are introduced in order to test the effect of linker on flexibility of luciferase activity. Glycine-rich linkers are introduced into P. pyralis firefly luciferase to make two separate mutant enzymes. Enzymatic properties of mutant and native forms were measured using luminescence assay. Results show that lengthening of luciferase domains through insertion of a flexible linker did not affect bioluminescence emission spectra. Also adding linkers do not have remarkable effect on thermostability. The Km values of mutants were increased compared to native form, indicating lower affinity of mutants toward substrates., (Copyright © 2016. Published by Elsevier B.V.)

We present here the effect of firefly luciferase surface charge saturation and the presence of some additives on its thermal-induced aggregation. Three mutants of firefly luciferase prepared by introduction of surface Arg residues named as 2R, 3R and 5R have two, three and five additional arginine residues substituted at their surface compared to native luciferase; respectively. Turbidimetric study of heat-induced aggregation indicates that all three mutants were reproducibly aggregated at higher rates relative to wild type in spite of their higher thermostability. Among them, 2R had most evaluated propensity to heat-induced aggregation. Therefore, the hydrophilization followed by appearing of more substituted arginine residues with positive charge on the firefly luciferase surface was not reduced its thermal aggregation. Nevertheless, at the same condition in the presence of charged amino acids, e.g. Arg, Lys and Glu, as well as a hydrophobic amino acid, e.g. Val, the heat-induced aggregation of wild type and mutants of firefly luciferases was markedly decelerated than those in the absence of additives. On the basis of obtained results it seems, relinquishment of variety in charge of amino acid side chains, they via local interactions with proteins cause to decrease rate and extent of their thermal aggregation., (© 2015 The American Society of Photobiology.)

Ionic liquids as neoteric solvents, microwave irradiation, and alternative energy source are becoming as a solvent for many enzymatic reactions. We recently showed that the incubation of firefly luciferase from Photinus pyralis with various ionic liquids increased the activity and stability of luciferase. Magnetic nanoparticles supported ionic liquids have been obtained by covalent bonding of ionic liquids-silane on magnetic silica nanoparticles. In the present study, the effects of [γ-Fe2O3@SiO2][BMImCl] and [γ-Fe2O3@SiO2][BMImI] were investigated on the structural properties and function of luciferase using circular dichroism, fluorescence spectroscopy, and bioluminescence assay. Enzyme activity and structural stability increased in the presence of magnetic nanoparticles supported ionic liquids. Furthermore, the effect of ingredients which were used was not considerable on K(m) value of luciferase for adenosine-5'-triphosphate and also K(m) value for luciferin.

Firefly bioluminescence reaction in the presence of Mg(2+), ATP and molecular oxygen is carried out by luciferase. The luciferase structure alterations or modifications of assay conditions determine the bioluminescence color of firefly luciferase. Among different beetle luciferases, Phrixothrix hirtus railroad worm emits either yellow or red bioluminescence color. Sequence alignment analysis shows that the red-emitter luciferase from Phrixothrix hirtus has an additional arginine residue at 353 that is absent in other firefly luciferases. It was reported that insertion of Arg in an important flexible loop350-359 showed changes in bioluminescence color from green to red and the optimum temperature activity was also increased. To explain the color tuning mechanism of firefly luciferase, the structure of native and a mutant (E354R/356R/H431Y) of Lampyris turkestanicus luciferase is determined at 2.7Å and 2.2Å resolutions, respectively. The comparison of structure of both types of Lampyris turkestanicus luciferases reveals that the conformation of this flexible loop is significantly changed by addition of two Arg in this region. Moreover, its surface accessibility is affected considerably and some ionic bonds are made by addition of two positive charge residues. Furthermore, we noticed that the hydrogen bonding pattern of His431 with the flexible loop is changed by replacing this residue with Tyr at this position. Juxtaposition of a flexible loop (residues 351-359) in firefly luciferase and corresponding ionic and hydrogen bonds are essential for color emission., (© 2013.)

Firefly luciferase catalyzes production of light from luciferin in the presence of Mg(2+)-ATP and oxygen. This enzyme has wide range of applications in biotechnology and development of biosensors. The low thermal stability of wild-type firefly luciferase is a limiting factor in most applications. Improvements in activity and stability of few enzymes in the presence of ionic liquids were shown in many reports. In this study, kinetic and thermal stability of firefly luciferase from Photinus pyralis in the presence of three tetramethylguanidine-based ionic liquids was investigated. The enzyme has shown improved activity in the presence of [1, 1, 3, 3-tetramethylguanidine][acetate], but in the presence of [TMG][trichloroacetate] and [TMG][triflouroacetate] activity, it decreased or unchanged significantly. Among these ionic liquids, only [TMG][Ac] has increased the thermal stability of luciferase. Incubation of [TMG][Ac] with firefly luciferase brought about with decrease of K(m) for ATP.

In order to better understand the functional role of an unusual residue (Thr346) of firefly luciferase mutagenesis at this residue was performed. Firefly luciferase, catalyzes the bioluminescence reaction and is an excellent tool as a reporter in nano-system biology studies. Nonetheless, the enzyme rapidly loses its activity at temperatures above 30 °C and this leads to reduced sensitivity and precision in analytical applications. Residue Thr346 in a connecting loop (341-348) of firefly luciferase is located in a disallowed region of Ramachandran plot. In this study, we have substituted this residue (T346) with anomalous dihedral angles with Val, Gly and Pro to clarify the role of this residue in structure and function of the enzyme using site-directed mutagenesis. Substitution of this unfavorable residue (T346) with atypical dihedral angles (ψ, φ) with other residues brought about an increase of thermostability and decrease of specific activity. Structural and functional properties of the mutants were analyzed using different spectroscopic methods. It seems that this residue is a critically conserved residue to support the functional flexibility for a fast kinetic bioluminescence reaction at the expense of lower stability., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Firefly luciferase is a protein with a large N-terminal and a small C-terminal domain. B-factor analysis shows that its C-terminal is much more flexible than its N-terminal. Studies on hyperthermophile proteins have been shown that the increased thermal stability of hyperthermophile proteins is due to their enhanced conformational rigidity and the relationship between flexibility, stability and function in most of proteins is on debate. Two mutations (D474K and D476N) in the most flexible region of firefly luciferase were designed. Thermostability analysis shows that D476N mutation doesn't have any significant effect but D474K mutation destabilized protein. On the other hand, flexibility analysis using dynamic quenching and limited proteolysis demonstrates that D474K mutation became much more flexible than wild type although D476N doesn't have any significant difference. Intrinsic and ANS fluorescence studies demonstrate that D476N mutation is brought about by structural changes without significant effect on thermostability and flexibility. Molecular modeling reveals that disruption of a salt bridge between D(474) and K(445) accompanying with some H-bond deletion may be involved in destabilization of D474K mutant., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Firefly luciferase-catalyzed reaction proceeds via the initial formation of an enzyme-bound luciferyl adenylate intermediate. The chemical origin of the color modulation in firefly bioluminescence has not been understood until recently. The presence of the same luciferin molecule, in combination with various mutated forms of luciferase, can emit light at slightly different wavelengths, ranging from red to yellow to green. A historical perspective of development in understanding of color emission mechanism is presented. To explain the variation in the color of the bioluminescence, different factors have been discussed and five hypotheses proposed for firefly bioluminescence color. On the basis of recent results, light-color modulation mechanism of firefly luciferase propose that the light emitter is the excited singlet state of OL(-) [(1)(OL(-))*], and light emission from (1)(OL(-))* is modulated by the polarity of the active-site environment at the phenol/phenolate terminal of the benzothiazole fragment in oxyluciferin.

Firefly luciferase catalyzes the oxidation of luciferin in the presence of ATP, Mg(2+) and molecular oxygen. The bioluminescence color of firefly luciferases is identified by the luciferase structure and assay conditions. Amongst different types of beetles, luciferase from Phrixotrix railroad worm (PhRE) with a unique additional residue (Arg353) naturally emits red bioluminescence color. By insertion of Arg356 in luciferase of Lampyris turkestanicus, corresponding to Arg353 in Phrixotrix hirtus, the color of the emitted light was changed to red. To understand the effect of this position on the bioluminescence color shift, four residues with similar sizes but different charges (Arg, Lys, Glu, and Gln) were inserted into Photinus pyralis luciferase. Comparison of mutants with native luciferase shows that mutation brought an increase in the content of secondary structure and globular compactness of (P. pylalis) luciferase. Comparative study of chemical denaturation of native and mutant luciferases by activity measurement, intrinsic and extrinsic fluorescence, circular dichroism, and DSC techniques revealed that insertion of positively charged residues (Arg, Lys) in the flexible loop (352-358) plays a significant role on the stability of (P. pyralis) luciferase and changes the light color to red.

Luciferases are the enzymes that catalyze the reactions that produce light in bioluminescence. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Amongst different beetle luciferases, those from phrixothrix rail-road worm with a unique additional residue (Arg353) emit red bioluminescence color naturally. Insertion of Arg(356) in Lampyris turkestanicus luciferase changed the emitted light to red with a bimodal bioluminescence spectrum. By insertion and substitution of positively-charged residues, different specific mutation (E354R/Arg(356), E354K/Arg(356), E354R, E354K) lead to changes of the bioluminescence color. Bioluminescence emission spectra indicate that substitution of E354 by R along with insertion of Arg(356) produces a luciferase that emits red light with a single peak bioluminescence spectrum. The comparison of mutants with native luciferase shows that mutations of firefly luciferase resulted in structural and functional thermostability. Comparative study of native and mutant luciferase (E354R/Arg(356)) by intrinsic and extrinsic fluorescence, CD spectropolarimetry, and homology modeling revealed mutation brought about an increase in content of secondary structure and globular compactness of L. turkestanicus luciferase. On the other hand, pK(a) of amino acids in the flexible loop decreased upon introducing of positive charges.

The effects of osmolytes, including sucrose, sorbitol and proline on the remaining activity of firefly luciferase were measured. Heat inactivation studies showed that these osmolytes maintain the remaining activity of enzyme and increase activation energy of thermal unfolding reaction. Fluorescence and circular dichroism (CD) experiments showed changes in secondary and tertiary structure of firefly luciferase, in the presence of sucrose, sorbitol and proline. The unfolding curves of luciferase (obtained by far-UV CD spectra), indicated an irreversible thermal denaturation and raising of the midpoint of the unfolding transition temperature (T(m)) in the presence of osmolytes.

The bioluminescence colours of firefly luciferases are determined by assay conditions and luciferase structure. Owing to red light having lower energy than green light and being less absorbed by biological tissues, red-emitting luciferases have been considered as useful reporters in imaging technology. A set of red-emitting mutants of Lampyris turkestanicus (Iranian firefly) luciferase has been made by site-directed mutagenesis. Among different beetle luciferases, those from Phrixothrix (railroad worm) emit either green or red bioluminescence colours naturally. By substitution of three specific amino acids using site-specific mutagenesis in a green-emitting luciferase (from L. turkestanicus), the colour of emitted light was changed to red concomitant with decreasing decay rate. Different specific mutations (H245N, S284T and H431Y) led to changes in the bioluminescence colour. Meanwhile, the luciferase reaction took place with relative retention of its basic kinetic properties such as K(m) and relative activity. Structural comparison of the native and mutant luciferases using intrinsic fluorescence, far-UV CD spectra and homology modelling revealed a significant conformational change in mutant forms. A change in the colour of emitted light indicates the critical role of these conserved residues in bioluminescence colour determination among firefly luciferases. Relatively high specific activity and emission of red light might make these mutants suitable as reporters for the study of gene expression and bioluminescence imaging.

Functional expression and spectroscopic analysis of luciferases from Lampyris turkestanicus and Photinus pyralis were carried out. cDNA encoding L. turkestanicus luciferase was isolated by reverse transcription-polymerase chain reaction, cloned, and functionally expressed in Escherichia coli. The luciferases were purified to homogeneity using Ni-nitrilotriacetic acid Sepharose, and kinetic properties of luciferase from L. turkestanicus were compared with that from P. pyralis. Amino acid differences in its primary structures in relation to P. pyralis luciferase brought about changes in the kinetic properties of the enzyme as evidenced by substantial lowering of Km for ATP, increased light decay time, and decreased thermostability. Luciferase from L. turkestanicus was used to carry out Michaelis-Menten kinetics with a Km of 95.5 muM for ATP and 20 muM for luciferin. Maximum activity was recorded at pH 8.5, so it might be a suitable reporter for microbial screening at alkaline pH. Tryptophan fluorescence for P. pyralis luciferase was higher than L. turkestanicus luciferase. Substitution of some residues in L. turkestanicus luciferase appears to change the kinetic properties by inducing a substantial tertiary structural change, without a large effect on secondary structural elements, as revealed by intrinsic and extrinsic fluorescence, Fourier transform infrared spectroscopy, and near-ultraviolet circular dichroism spectra.

The cDNA of a firefly luciferase from lantern mRNA of Lampyroidea maculata has been cloned, sequenced and functionally expressed. The cDNA has an open reading frame of 1647 bp and codes for a 548-residue-long polypeptide. Noteworthy, sequence comparison as well as homology modeling showed the highest degree of similarity with H. unmunsana and L. mingrelica luciferases, suggesting a close phylogenetic relationship despite the geographical distance separation. The deduced amino acid sequence of the luciferase gene of firefly L. maculata showed 93% identity to H. unmunsana. Superposition of the three-dimensional model of L. maculata luciferase (generated by homology modeling) and three dimensional structure of Photinus pyralis luciferase revealed that the spatial arrangements of Luciferin and ATP-binding residues are very similar. Putative signature of AMPbinding domain among the various firefly species and Lampyroidea maculata was compared and a striking similarity was found. Different motifs and sites have been identified in Lampyroidea maculata by sequence analysis. Expression and purification of luciferase from Lampyroidea maculata was carried out using Ni-NTA Sepharose. Bioluminescence emission spectrum was similar to Photinus pyralis luciferase.

The first cDNA from lampyridae encoding a glow-worm luciferase from lantern mRNA of Lampyris turkestanicus has been cloned, sequenced, the amino acid sequence predicted, and the sequence reported to GenBank. The cDNA was 1644 base pairs in length and coding a 547-residue protein. The deduced amino acid sequence of the luciferase gene of L. turkestanicus showed 98.7% and 95.8% identity to Lampyris noctiluca and Pyrocoelia rufa, respectively. Phylogenetic analysis further confirmed that the deduced amino acid sequences of L. turkestanicus luciferase gene belong to the same subfamily, Lampyrinae. The cDNA encoding the luciferase of L. turkestanicus was expressed as a 62kDa band in recombinant Escherichia coli and showed green luminescence in the presence of luciferin. Amongst amino acid differences of L. turkestanicus and L. noctiluca (its clade) there are two important substitutions. Signature amino-acid sequences and motifs found in the deduced sequence are CK2-phospho site, ASN-glycosylation, myristoylation site, PKC-phospho site, microbodies C-terminal targeting signal, and AMP-binding domain.

Objective(s): Firefly luciferase is a monooxygenase enzyme that emits flash of light during the enzymatic reaction. Luciferase has been used in many bioanalytical fields from ATP detection methods to in vivo imaging. In recent decades, focus has been carried out on nanoparticles for their fluorescence properties. Semiconductor quantum dots have unique tunable properties that turn them promising tools in biological and biomedical researches, as nanosensors, photo-electrochemical and light-emitting devices. Carbon-based nanoparticles such as graphene quantum dots (GQDs) have useful benefits such as low toxicity, suitable luminescence and easy preparation. Materials and Methods: In ths study, recombinant p. pyralis luciferase was expressed and purified based on N-terminal His-tag and then kinetic parameters of enzyme activity such as Km and Vmax values in presence and absence of GQDs were calculated. Results: The results showed that Km for ATP andluciferin substrates in the presence of GQDs were increased. Fluorescence spectroscopy showed significant changes in protein structure or in fluorescence spectra and decrease in the activity of the luciferase in presence of GQD. Both loss of activity and increase of substrates Km showed decrease of catalytic efficiency presumably through structural alteration. Conclusion: From these data it can be concluded that the protein structure under the influence of GQD may have changed that lead to alteration of enzyme activity. [ABSTRACT FROM AUTHOR]

Abstract: Stabilization and immobilization on a proper support is essential in processing and production of enzymes. Luciferase is an unstable, thermally sensitive, and light-emitting enzyme that requires stabilization for efficiency and productivity in bio-luminescence reactions. In this work, a known and well-porous framework, MIL-53(Al), was used, for the first time, to support the immobilization of firefly luciferase. Interestingly, all the stability values were improved through the immobilization process, and the content of the loading enzyme on MIL-53(Al) was found to be significant. The results obtained indicated that the stabilization of firefly luciferase on MIL-53(Al) proceeded through the adsorption method. MIL-53(Al) was also characterized by the FT-IR, X-ray diffraction, FE-SEM, TEM, BET, and TGA techniques in order to determine the carboxylic groups and symmetric/asymmetric CO2, percentage of crystallinity and its purity, morphology, size, and shape of the MIL-53(Al) particles, and surface-to-area ratio and thermal gravimetry of support.Graphical abstract: [ABSTRACT FROM AUTHOR]

Luciferase is the key component of light production in bioluminescence process. Extensive and advantageous application of this enzyme in biotechnology is restricted due to its low thermal stability. Here we report the effect of heating up above Tm on the structure and dynamical properties of luciferase enzyme compared to temperature at 298 K. In this way we demonstrate that the number of hydrogen bonds between N- and C-domain is increased for the free enzyme at 325 K. Increased inter domain hydrogen bonds by three at 325 K suggests that inter domain contact is strengthened. The appearance of simultaneous strong salt bridge and hydrogen bond between K529 and D422 and increased existence probability between R533 and E389 could mechanistically explain stronger contact between N- and C-domain. Mutagenesis studies demonstrated the importance of K529 and D422 experimentally. Also the significant reduction in SASA for experimentally important residues K529, D422 and T343 which are involved in active site region was observed. Principle component analysis (PCA) in our study shows that the dynamical behavior of the enzyme is changed upon heating up which mainly originated from the change of motion modes and associated extent of those motions with respect to 298 K. These findings could explain why heating up of the enzyme or thermal fluctuation of protein conformation reduces luciferase activity in course of time as a possible mechanism of thermal functional inactivation. According to these results we proposed two strategies to improve thermal stability of functional luciferase. [ABSTRACT FROM AUTHOR]

Protein engineering can provide useful approaches for loop anchoring and mutation of surface-exposed loop residues to Arg for the design of thermostable proteins. In this context and due to the high proportion of surface loops, some of the solvent-exposed residues in the Lampyris turkestanicus luciferase were mutated to Arg. Using the red-emitter mutant luciferase (E354R/Arg356), the single (-Q35R, -I182R, -I232R and -L300R), double (-Q35R/I232R) and triple (-Q35R/I232R/I182R) mutant luciferases were introduced. The relative remaining activity of -I232R, double and triple mutants increased significantly compared to the wild-type at 40 °C. The optimal temperature of these mutants increased up to 40 °C which were 15 °C more than wild-type luciferase. It is anticipated that these mutations increased the local interactions that finally improved the thermostability and optimum temperature of luciferase. It should be noted that Arg substitution at amino acid positions 35, 182 and 232 had no effect on the bioluminescence emission spectra. Furthermore, these mutations have not significantly changed the specific activities of firefly luciferases. Finally, with the use of the homology modeling and molecular docking, the effects of these substitutions were evaluated. In conclusion, this study provides beneficial insights on how the thermal stability of luciferase can be improved by protein engineering for biological applications. [ABSTRACT FROM AUTHOR]

Replacement of some hydrophobic solvent-exposed residues in Lampyris turkestanicus luciferase with arginine increases thermostability of this enzyme. Herein, thermodynamic and kinetic of unfolding reactions of wild type (WT), E354R/356R, E354R/356R-I232R and E354R/356R-Q35R/L182R/I232R variants, has been investigated. Fluorescence and Far-UV circular dichroism measurements using urea as a chemical denaturant indicated that the value of [ABSTRACT FROM AUTHOR]

The effects of mobile phone frequency electromagnetic field (RF-EMF, 940 MHz) on a stable cell line (HEK293T) harbouring the firefly luciferase gene were evaluated. A waveguide exposure system with 1 W input power provided the mean specific absorption rate of ≈0.09 W kg−1 in 35 mm Petri dishes. The effects of exposure duration (15, 30, 45, 60 and 90 min) on luciferase activity and oxidative response elements were investigated. Endogenous luciferase activity was reduced after 30 and 45 min of continuous exposure, while after 60 min, the exposed cell lysate showed higher luciferase activity compared with the non-exposed control. Reactive oxygen species (ROS) generation was highest in the 30 min exposed cells as studied by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence. The observed boost in ROS was then followed by a sharp rise in catalase (CAT) and superoxide dismutase (SOD) activity and elevation of glutathione (GSH) during the 45 min exposure. Decrease in lipid peroxidation (malondialdehyde, MDA) was meaningful for the 45 and 60 min exposed cells. Therefore, it appears that an increase in the activity of luciferase after 60 min of continuous exposure could be associated with a decrease in ROS level caused by activation of the oxidative response. This ability in cells to overcome oxidative stress and compensate the luciferase activity could also be responsible for the adaptive response mechanism detected in ionizing radiation studies with RF-EMF pre-treatments. [ABSTRACT FROM AUTHOR]

Abstract: Possible roles of two different Arginine (Arg; R) 213 and 337 on kinetic and structural stability of Photinus pyralis luciferase have been investigated using thermal and chemical denaturation studies. This enzyme is highly sensitive to protease digestion and temperature, which limits its fieldability, particularly for in vivo imaging. In order to generate more stable luciferases against trypsin digestion, site-directed mutagenesis was conducted to block two representative tryptic sites on the surface of N-terminal domain, via substitution of Arg213 and Arg337 by methionine (Met; M) and glutamine (Gln; Q), respectively [A. Riahi-Madvar, S. Hosseinkhani, Protein engineering, design and selection 22 (2009) 655–663]. The improvement of mutant enzymes stability against protease hydrolysis may be attributed to the more rigidity of the enzyme structure upon mutations, as can be deducted from elevated levels of m U-N values and decrease of activation energy. Furthermore, mutation at position 337 which is accompanied with more alteration on the basic kinetic properties relative to mutation at position 213, revealed the high values of the , half-time of inactivation at 30°C and T m for R337Q where Arg213 is maintained in structure. Based on the results, it can be concluded that whilst Arg213 affects structural stability, Arg337 is critical for kinetic stability. [Copyright &y& Elsevier]

The importance and applicability of ionic liquids (ILs) in biocatalysis have been well recognized. ILs have growing interest as new and highly efficient reaction mediums for biocatalytic reactions, but as a reaction milieu for firefly luciferase has not been tested. In this report, the effects of two tetramethylguanidine-based ionic liquids on the activity and stability of Photinus pyralis luciferase were investigated. In spite of a common cationic part, luciferase activity increased up to 0.25 M of [TMG][Lac] but decreased in the presence of similar concentrations of [TMG][Pro]. Optimum temperature and thermal stability studies show more stability of luciferase only in the presence of [TMG][Lac]. The change in light intensity of firefly luciferase in the presence of both ILs was brought about without effect on bioluminescence emission spectra. The rate of light decay in the presence of both ILs was slower than native luciferase. [ABSTRACT FROM AUTHOR]

The thermal sensitivity and pH-sensitive spectral properties of firefly luciferase have hampered its application in a variety of fields. It is proposed that the stability of a protein can be increased by introduction of disulfide bridge that decreases the configurational entropy of unfolding. A disulfide bridge is introduced into Photinus pyralisfirefly luciferase to make two separate mutant enzymes with a single bridge. Even though the A103C/S121C mutant showed remarkable thermal stability, its specific activity decreased, whereas the A296C/A326C mutant showed tremendous thermal stability, relative pH insensitivity and 7.3-fold increase of specific activity. Moreover, the bioluminescence emission spectrum of A296C/A326C was resistant against higher temperatures (37 °C). Far-UV CD analysis showed slight secondary structure changes for both mutants. Thermal denaturation analysis showed that conformational stabilities of A103C/S121C and A296C/A326C are more than native firefly luciferase. It is proposed that since A296 and A326 are situated in the vicinity of the enzyme active site microenvironment in comparison with A103 and S121, the formation of a disulfide bridge in this region has more impact on enzyme kinetic characteristics. [ABSTRACT FROM AUTHOR]

Firefly luciferase (EC.1.13.12.7) from Photinus pyralis is a single polypeptide chain (62 kDa), responsible for emission of yellow-green (557 nm) light, known to be most efficient bioluminescence system that make it an excellent tool for reporter in nano-system biology. However, it is very sensitive to proteolytic degradation, which reduces its intracellular half-life, leads to loss in sensitivity and precision in analytic applications. In order to generate more stable luciferases against protease digestion, we substituted two tryptic sites: R213, R337 and also next residue to it (Q338) with another amino acids. Overall, all mutations brought about structural changes that indicated more compact structure upon mutation, which revealed by enhancement of tryptophan fluorescence, decreases flexibility and less surface hydrophobic pockets. In general, structural changes associated with a clear improvement in thermostability and resistance against trypsin hydrolysis. In particular, R337Q mutant shows higher light stability in mammalian cell culture, which makes it as a suitable reporter for imaging. [ABSTRACT FROM PUBLISHER]

Firefly luciferase is a monooxygenase that catalyzes the ATP-dependent conversion of firefly luciferin into a luciferyl-adenylate, which is oxidized to an electronically excited oxyluciferin in a multistep reaction and produces visible light with a remarkable quantum yield. [he bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Among different beetles, only luciferase from Phrixotrix railroad worm (PhRE) emits red bioluminescence, naturally. The presence of Arg353 in P/IRE luciferase, which corresponds to the deleted residue in the other luciferases, is an important distinctive structural feature of it. Insertion of Arg356 into a green-emitter luciferase (Lampyris turkestanicus), corresponding to Arg353 in Phrixotrix hirtus, changed the emitted light from green to red. To further clarify the effect of this position on the light shift mechanism, four residues with similar sizes but different charges (Arg, Lys, Glu, and Gin) were inserted into Photinus pyralis luciferase, using site-specific insertion mutagenesis. Insertion of a residue with a positive side chain (Arg356 and Lys356) changed the light color to red, while insertion of a residue with a negative side chain (Glu356) had little effect on color. Insertion of a neutral residue (Glu356) at this position was performed without any change in bioluminescence spectra. Insertion of positively charged residues in this loop took place with a series of structural changes which were confirmed by fluorescence spectroscopy and homology modeling. Homology modeling reveals the appearance of a bulge in a flexible loop (T352-P359) upon mutation which shifts to the left side with a color change from green to red. [ABSTRACT FROM AUTHOR]

• Mutations of two residues did not effect in bioluminescence color shift. • Km values for luciferin were decreased in both mutants comparing to wild type. • Rate of activity decrease in both mutations were increased compared to wild type. • Mutation brought notable changes in secondary and tertiary structures. Firefly luciferase as a bioluminescent enzyme has many applications in various fields from scientific research to commercial goals. This enzyme is relatively unstable with low functional capacity due to rapid inactivation in physiological temperature, low in vitro stability and high susceptibility to proteolytic degradation. Based on previous studies, two regions 206–220 and 329–341 on N-domain of Photinus pyralis luciferase are known accessible and flexible. Flexible regions may lead to protein instability. Here, the effect of mutation at positively charged residues Lys(K)329 and Arg(R)330 on the stability of luciferase was studied. Furthermore, the role of these mutations on the structure and function was evaluated. Introducing of these point mutations did not affect the orientation of critical residues in bioluminescence color determination. The kinetic studies showed that thermostability and Km value for luciferin in both mutants were decreased as compared to wild type. However, optimum pH and optimum temperature showed no significant changes in both mutants. Moreover, the structural data revealed an increase in tryptophan fluorescence intensity and secondary structure content for R330Q in compared with wild type, while intrinsic fluorescence and far-UV CD intensity in K329I mutant was decreased. [ABSTRACT FROM AUTHOR]