Site-Directed Mutagenesis of Histidine 245 in Firefly Luciferase:  A Proposed Model of the Active Site

Abstract

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.

Active-Site Properties of Phrixotrix Railroad Worm Green and Red Bioluminescence-Eliciting Luciferases

“…Luciferin-Binding Sites of Phrixotrix LuciferasesAccording to Branchini et al (13) and Ugarova (14), the luciferin-binding site in P. pyralis firefly luciferase involves residues R218, 244HHGF247, 315GG316, 340YGLTE344, A348, and I351. In the Phrixotrix luciferases, these positions correspond to R215, 241HHAF245, 312GGA(S)314, 337YGLTE341, A345, and L348, respectively (29).…”

“…However, the physicochemical properties of only a few of them have been studied in detail (10,11). The three-dimensional structure of Photinus pyralis firefly luciferase was resolved in the absence of substrates (12), and two active-site models have been proposed (13,14). According to these models, the luciferin-binding site consists of residues R218, 244HHGF247, 315GG316, R330, T343, S347, A348, and I351.…”

Enhanced red‐emitting railroad worm luciferase for bioassays and bioimaging

“…Such a notable increase in activity for I212L/N351K may be explained by the combined mutations of I212L and N351K, which might cause a local change in the microenvironment of the active site. A previously proposed active site model 38 will support that the replacement of N351 of PhRED affect the structure of the active site 39 for changing stability and activity, because N351 of PhRED corresponds to E354 of Ppy, which locates around the ATPbinding pocket (comprising 316-318, 339-342, and 362) and the luciferin-binding pocket (surrounded by 245-251, 315-317, 341-343, and 346-348). I212L/N351K and N351K showed consistently higher NIH3T3 cell-based activities than did WT and other mutants, whereas I212L, S463R, and I212L/ S463R had lower cell-based activities than WT [ Fig.…”

“…51 Unlike N351K in this study, mutations of the corresponding positions in other green-yellow-emitting firefly luciferases caused a small red shift. 32,38 Materials and Methods…”

Mammalian-Based Bioreporter Targets: Protein Expression for Bioluminescent and Fluorescent Detection in the Mammalian Cellular Background

“…Despite the widespread use of Luc, the actual binding sites for these components have yet to be determined conclusively. There are two current models put forth by Ugarova (Sandalova & Ugarova, 1999) and Branchini (Branchini, B. et al, 1998) that propose similar active site interactions. Both models suggest that residues Arg 218, His 245-Phe 247, Ala 313-Gly 320, and Lys 529 form the binding site for D-luciferin, with a hydrophobic surface being contributed directly by Ala 313, Ala 348, Ile 351, and Phe 247.…”