Glu311 and Arg337 Stabilize a Closed Active-site Conformation and Provide a Critical Catalytic Base and Countercation for Green Bioluminescence in Beetle Luciferases.

Beetle luciferases elicit the emission of different bioluminescence colors from green to red. Whereas firefly luciferases emit yellow-green light and are pH-sensitive, undergoing a typical red-shift at acidic pH and higher temperatures and in the presence of divalent heavy metals, click beetle and railroadworm luciferases emit a wider range of colors from green to red but are pH-independent. Despite many decades of study, the structural determinants and mechanisms of bioluminescence colors and pH sensitivity remain enigmatic. Here, through modeling studies, site-directed mutagenesis, and spectral and kinetic studies using recombinant luciferases from the three main families of bioluminescent beetles that emit different colors of light (Macrolampis sp2 firefly, Phrixotrix hirtus railroadworm, and Pyrearinus termitilluminans click beetle), we investigated the role of E311 and R337 in bioluminescence color determination. All mutations of these residues in firefly luciferase produced red mutants, indicating that the preservation of opposite charges and the lengths of the side chains of E311 and R337 are essential for keeping a salt bridge that stabilizes a closed hydrophobic conformation favorable for green light emission. Kinetic studies indicate that residue R337 is important for binding luciferin and creating a positively charged environment around excited oxyluciferin phenolate. In Pyrearinus green-emitting luciferase, the R334A mutation causes a 27 nm red-shift, whereas in Phrixotrix red-emitting luciferase, the L334R mutation causes a blue-shift that is no longer affected by guanidine. These results provide compelling evidence that the presence of arginine at position 334 is essential for blue-shifting the emission spectra of most beetle luciferases. Therefore, residues E311 and R337 play both structural and catalytic roles in bioluminescence color determination, by stabilizing a closed hydrophobic conformation favorable for green light emission, and also providing a base to accept excited oxyluciferin phenol proton, and a countercation to shield the negative charge of E311 and to stabilize excited oxyluciferin phenolate, blue-shifting emission spectra in most beetle luciferases.

Molecular phylogeny of Neotropical bioluminescent beetles (Coleoptera: Elateroidea) in southern and central Brazil.

Bioluminescence in beetles is found mainly in the Elateroidea superfamily (Elateridae, Lampyridae and Phengodidae). The Neotropical region accounts for the richest diversity of bioluminescent species in the world with about 500 described species, most occurring in the Amazon, Atlantic rainforest and Cerrado (savanna) ecosystems in Brazil. The origin and evolution of bioluminescence, as well as the taxonomic status of several Neotropical taxa in these families remains unclear. In order to contribute to a better understanding of the phylogeny and evolution of bioluminescent Elateroidea we sequenced and analyzed sequences of mitochondrial NADH2 and the nuclear 28S genes and of the cloned luciferase sequences of Brazilian species belonging to the following genera: (Lampyridae) Macrolampis, Photuris, Amydetes, Bicellonycha, Aspisoma, Lucidota, Cratomorphus; (Elateridae) Conoderus, Pyrophorus, Hapsodrilus, Pyrearinus, Fulgeochlizus; and (Phengodidae) Pseudophengodes, Phrixothrix, Euryopa and Brasilocerus. Our study supports a closer phylogenetic relationship between Elateridae and Phengodidae as other molecular studies, in contrast with previous morphologic and molecular studies that clustered Lampyridae/Phengodidae. Molecular data also supported division of the Phengodinae subfamily into the tribes Phengodini and Mastinocerini. The position of the genus Amydetes supports the status of the Amydetinae as a subfamily. The genus Euryopa is included in the Mastinocerini tribe within the Phengodinae/Phengodidae.

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.

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.

Mitochondrial genome sequence of the Brazilian luminescent click beetle Pyrophorus divergens (Coleoptera: Elateridae): mitochondrial genes utility to investigate the evolutionary history of Coleoptera and its bioluminescence.

The Coleoptera order is the richest group among Metazoa, but its phylogenetics remains incompletely understood. Among Coleoptera, bioluminescence is found within the Elateroidea, but the evolution of this character remains a mystery. Mitochondrial DNA has been used extensively to reconstruct phylogenetic relationships, however, the evolution of a single gene does not always correspond to the species evolutionary history and the molecular marker choice is a key step in this type of analysis. To create a solid basis to better understand the evolutionary history of Coleoptera and its bioluminescence, we sequenced and comparatively analyzed the mitochondrial genome of the Brazilian luminescent click beetle Pyrophorus divergens (Coleoptera: Elateridae).

Active-site properties of Phrixotrix railroad worm green and red bioluminescence-eliciting luciferases.

The luciferases of the railroad worm Phrixotrix (Coleoptera: Phengodidae) are the only beetle luciferases that naturally produce true red bioluminescence. Previously, we cloned the green- (PxGR) and red-emitting (PxRE) luciferases of railroad worms Phrixotrix viviani and P. hirtus[OLE1]. These luciferases were expressed and purified, and their active-site properties were determined. The red-emitting PxRE luciferase displays flash-like kinetics, whereas PxGR luciferase displays slow-type kinetics. The substrate affinities and catalytic efficiency of PxRE luciferase are also higher than those of PxGR luciferase. Fluorescence studies with 8-anilino-1-naphthalene sulfonic acid and 6-p-toluidino-2-naphthalene sulfonic acid showed that the PxRE luciferase luciferin-binding site is more polar than that of PxGR luciferase, and it is sensitive to guanidine. Mutagenesis and modelling studies suggest that several invariant residues in the putative luciferin-binding site of PxRE luciferase cannot interact with excited oxyluciferin. These results suggest that one portion of the luciferin-binding site of the red-emitting luciferase is tighter than that of PxGR luciferase, whereas the other portion could be more open and polar.

A new firefly luciferase with bimodal spectrum: identification of structural determinants of spectral pH-sensitivity in firefly luciferases.

Fireflies emit flashes in the green-yellow region of the spectrum for the purpose of sexual attraction. The bioluminescence color is determined by the luciferases. It is well known that the in vitro bioluminescence color of firefly luciferases can be shifted toward the red by lower pH and higher temperature; for this reason they are classified as pH-sensitive luciferases. However, the mechanism and structural origin of pH sensitivity in fireflies remains unknown. Here we report the cloning of a new luciferase from the Brazilian twilight active firefly Macrolampis sp2, which displays an unusual bimodal spectrum. The recombinant luciferase displays a sensitive spectrum with the peak at 569 nm and a shoulder in the red region. Comparison of the bioluminescence spectra of Macrolampis, Photinus and Cratomorphus firefly luciferases shows that the distinct colors are determined by the ratio between green and red emitters under luciferase influence. Comparison of Macrolampis luciferase with the highly similar North American Photinus pyralis luciferase (91%) showed few substitutions potentially involved with the higher spectral sensitivity in Macrolampis luciferase. Site-directed mutagenesis showed that the natural substitution E354N determines the appearance of the shoulder in the red region of Macrolampis luciferase bioluminescence spectrum, helping to identify important interactions and residues involved in the pH-sensing mechanism in firefly luciferases.

The structural origin and biological function of pH-sensitivity in firefly luciferases.

Firefly luciferases are called pH-sensitive because their bioluminescence spectra display a typical red-shift at acidic pH, higher temperatures, and in the presence of heavy metal cations, whereas other beetle luciferases (click beetles and railroadworms) do not, and for this reason they are called pH-insensitive. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. This subject is revised in view of recent results. Some substitutions of amino-acid residues influencing pH-sensitivity in firefly luciferases have been identified. Sequence comparison, site-directed mutagenesis and modeling studies have shown a set of residues differing between pH-sensitive and pH-insensitive luciferases which affect bioluminescence colors. Some substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). A network of hydrogen bonds and salt bridges involving the residues N229-S284-E311-R337 was found to be important for affecting bioluminescence colors. It is suggested that these structural elements may affect the benzothiazolyl side of the luciferin-binding site affecting bioluminescence colors. Experimental evidence suggest that the residual red light emission in pH-sensitive luciferases could be a vestige that may have biological importance in some firefly species. Furthermore, the potential utility of pH-sensitivity for intracellular biosensing applications is considered.