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.

The role of lysine 529, a conserved residue of the acyl-adenylate-forming enzyme superfamily, in firefly luciferase.

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from the substrates luciferin, Mg-ATP, and oxygen in a two-step process. The enzyme first catalyzes the adenylation of the carboxylate substrate luciferin with Mg-ATP followed by the oxidation of the acyl-adenylate to the light-emitting oxyluciferin product. The beetle luciferases are members of a large family of nonbioluminescent proteins that catalyze reactions of ATP with carboxylate substrates to form acyl-adenylates. Formation of the luciferase-luciferyl-AMP complex is a specific example of the chemistry common to this enzyme family. Site-directed mutants at positions Lys529, Thr343, and His245 were studied to determine the effects of the amino acid changes at these positions on the individual luciferase-catalyzed adenylation and oxidation reactions. The results suggest that Lys529 is a critical residue for effective substrate orientation and that it provides favorable polar interactions important for transition state stabilization leading to efficient adenylate production. These findings as well as those with the Thr343 and His245 mutants are interpreted in the context of the firefly luciferase X-ray structures and computational-based models of the active site.

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.

Site-directed mutagenesis of histidine 245 in firefly luciferase: a proposed model of the active site.

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate luciferin by a sequence of reactions that require Mg-ATP and molecular oxygen. We previously reported [Branchini, B. R., Magyar, R. A., Marcantonio, K. M., Newberry, K. J., Stroh, J. G., Hinz, L. K., and Murtiashaw, M. H. (1997) J. Biol. Chem. 272, 19359-19364] that 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a firefly luciferin analogue, was a potent photoinactivation reagent for luciferase. We identified a luciferase peptide 244HHGF247, the degradation of which was directly correlated to the photooxidation process. We report here the construction and purification of wild-type and mutant luciferases H244F, H245F, H245A, and H245D. The results of photoinactivation and kinetic and bioluminescence studies with these proteins are consistent with His245 being the primary functional target of BPTC-catalyzed enzyme inactivation. The possibility that His245 is oxidized to aspartate during the photooxidation reaction was supported by the extremely low specific activity ( approximately 300-fold lower than WT) of the H245D mutant. Using the crystal structures of luciferase without substrates [Conti, E., Franks, N. P., and Brick, P. (1996) Structure 4, 287-298] and the functionally related phenylalanine-activating subunit of gramicidin synthetase 1 [Conti, E., Stachelhaus, T., Marahiel, M. A., and Brick, P. (1997) EMBO J. 16, 4174-4183] as a starting point, we have performed molecular-modeling studies and propose here a model for the luciferase active site with substrates luciferin and Mg-ATP bound. We have used this model to provide a structure-based interpretation of the role of 244HHGF247 in firefly bioluminescence.

Identification of a firefly luciferase active site peptide using a benzophenone-based photooxidation reagent.

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate luciferin by a series of reactions that require MgATP and molecular oxygen. We prepared 2-(4-benzoylphenyl)thiazole-4-carboxylic acid (BPTC), a novel benzophenone-based substrate analog, intending to use it in photoaffinity labeling studies to probe the luciferase active site. Instead, we found that while BPTC was a potent photoinactivating reagent for firefly luciferase, it was not a photoaffinity labeling agent. Using proteolysis, reverse phase high-performance liquid chromatography, tandem high performance liquid chromatography-electrospray ionization mass spectrometry, and Edman sequencing, we identified a single luciferase peptide, 244HHGF247, the degradation of which was directly correlated to luciferase photoinactivation. Results of enzyme kinetics and related studies were consistent with this peptide being at or near the luciferin binding site. Further, peptide model studies and additional investigations on the nature of the photoinactivation process strongly suggested that BPTC catalyzed the formation of singlet oxygen at the active site of the enzyme. We describe here an uncommon example of active site-directed photooxidation of an enzyme by singlet oxygen.

Mutation of a protease-sensitive region in firefly luciferase alters light emission properties.

Luciferase (EC 1.13.12.7) from the North American firefly, Photinus pyralis, is widely used as a reporter enzyme in cell biology. One of its distinctive properties is a pronounced susceptibility to proteolytic degradation that causes luciferase to have a very short intracellular half-life. To define the structural basis for this behavior and possibly facilitate the design of more stable forms of luciferase, limited proteolysis studies were undertaken using trypsin and chymotrypsin to identify regions of the protein whose accessible and flexible character rendered them especially sensitive to cleavage. Regions of amino acid sequence 206-220 and 329-341 were found to be sensitive, and because the region around 206-220 had high homology with other luciferases, CoA ligases, and peptidyl synthetases, this region was selected for mutagenesis experiments intended to determine which of its amino acids were essential for activity. Surprisingly, many highly conserved residues including Ser198, Ser201, Thr202, and Gly203 could be mutated with little effect on the luminescent activity of P. pyralis luciferase. One mutation, however, S198T, caused several alterations in enzymatic properties including shifting the pH optimum from 8.1 to 8.7, lowering the Km for Mg-ATP by a factor of 4 and increasing the half-time for light emission decay by a factor of up to 150. While the S198T luciferase was less active than wild type, activity could be restored by the introduction of the additional L194F and N197Y mutations. In addition to indicating the involvement of this region in ATP binding, these results provide a new form of the enzyme that affords a more versatile reporter system.

Inactivation of firefly luciferase with N-(iodoacetyl)-N'- (5-sulfo-1-naphthyl)ethylenediamine (I-AEDANS).

N-Iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (I-AEDANS), a fluorescent reagent that selectively modifies cysteine residues, was demonstrated to irreversibly inhibit native Photinus pyralis luciferase purified from firefly lanterns. Complete inactivation of luciferase activity was accompanied by the blockage of all four cysteine thiols and the concomitant incorporation of 4 mol of N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS) per mole of enzyme. Employing proteolytic digestions of AEDANS-labeled luciferase and reverse-phase-high-performance liquid chromatography (RP-HPLC), seven tagged peptides were isolated. The AEDANS label provided a convenient spectroscopic marker for the identification of the modified peptides. The sequences of the labeled peptides were deduced from electrospray ionization mass spectrometry (ESMS) and N-terminal sequencing. The fluorescent peptides included cysteine residues and spanned sequences composed of amino acids Leu78-Lys85, Thr214-Arg218, Asp224-Arg275, and Gly388-Met396. The luciferin substrate provided substantial protection against luciferase inactivation resulting in a 60-67% decrease in the labeling of all four cysteine thiols. Thus, it does not appear that a specific cysteine mediates the loss of luciferase activity. Additional LC/ESMS studies permitted the identification of 78% of the native luciferase molecule, which, unlike the recombinant protein, was found to contain an acetylated N-terminus. The AEDANS labeling results and the identification of well-defined proteolytic fragments should facilitate future structure-function investigations of the firefly luciferases.