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Structural basis for the spectral difference in luciferase bioluminescence


Fireflies communicate with each other by emitting yellow-green to yellow-orange brilliant light. The bioluminescence reaction, which uses luciferin, Mg-ATP and molecular oxygen to yield an electronically excited oxyluciferin species, is carried out by the enzyme luciferase. Visible light is emitted during relaxation of excited oxyluciferin to its ground state. The high quantum yield1 of the luciferin/luciferase reaction and the change in bioluminescence colour caused by subtle structural differences in luciferase have attracted much research interest. In fact, a single amino acid substitution in luciferase changes the emission colour from yellow-green to red2,3,4,5. Although the crystal structure of luciferase from the North American firefly (Photinus pyralis) has been described6,7, the detailed mechanism for the bioluminescence colour change is still unclear8,9,10,11. Here we report the crystal structures of wild-type and red mutant (S286N) luciferases from the Japanese Genji-botaru (Luciola cruciata) in complex with a high-energy intermediate analogue, 5′-O-[N-(dehydroluciferyl)-sulfamoyl]adenosine (DLSA). Comparing these structures to those of the wild-type luciferase complexed with AMP plus oxyluciferin (products) reveals a significant conformational change in the wild-type enzyme but not in the red mutant. This conformational change involves movement of the hydrophobic side chain of Ile 288 towards the benzothiazole ring of DLSA. Our results indicate that the degree of molecular rigidity of the excited state of oxyluciferin, which is controlled by a transient movement of Ile 288, determines the colour of bioluminescence during the emission reaction.

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Figure 1: The bioluminescence reaction catalysed by luciferase.
Figure 2: Structure of Lcr luciferase·DLSA complex and DLSA binding site.
Figure 3: Comparison of the luciferin binding site in the Lcr luciferase structures.
Figure 4: Bioluminescence colour of wild-type and three mutant forms (I288V, I288A and S286N) of Lcr luciferases.


  1. Seliger, H. H. & McElroy, W. D. Spectral emission and quantum yield of firefly bioluminescence. Arch. Biochem. Biophys. 88, 136–141 (1960)

    Article  CAS  Google Scholar 

  2. Kajiyama, N. & Nakano, E. Isolation and characterization of mutants of firefly luciferase which produce different colors of light. Protein Eng. 4, 691–693 (1991)

    Article  CAS  Google Scholar 

  3. Mamaev, S. V., Laikhter, A. L., Arslan, T. & Hecht, S. M. Firefly luciferase: Alteration of the color of emitted of light resulting from substitutions at position 286. J. Am. Chem. Soc. 118, 7243–7244 (1996)

    Article  CAS  Google Scholar 

  4. Branchini, B. R., Southworth, T. L., Murtiashaw, M. H., Boije, H. & Fleet, S. E. A mutagenesis study of the putative luciferin binding site residues of firefly luciferase. Biochemistry 42, 10429–10436 (2003)

    Article  CAS  Google Scholar 

  5. Branchini, B. R. et al. Site-directed mutagenesis of firefly luciferase active site amino acids: a proposed model for bioluminescence color. Biochemistry 38, 13223–13230 (1999)

    Article  CAS  Google Scholar 

  6. Conti, E., Franks, N. P. & Brick, P. Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure 4, 287–298 (1996)

    Article  CAS  Google Scholar 

  7. Franks, N. P., Jenkins, A., Conti, E., Lieb, W. R. & Brick, P. Structural basis for the inhibition of firefly luciferase by a general anesthetic. Biophys. J. 75, 2205–2211 (1998)

    Article  CAS  Google Scholar 

  8. DeLuca, M. Hydrophobic nature of the active site of firefly luciferase. Biochemistry 8, 160–166 (1969)

    Article  CAS  Google Scholar 

  9. White, E. H., Rapaport, E., Hopkins, T. A. & Seliger, H. H. Chemi- and bioluminescence of firefly luciferin. J. Am. Chem. Soc. 91, 2178–2180 (1969)

    Article  CAS  Google Scholar 

  10. Branchini, B. R. et al. An alternative mechanism of bioluminescence color determination in firefly luciferase. Biochemistry 43, 7255–7262 (2004)

    Article  CAS  Google Scholar 

  11. McCapra, F., Gilfoyle, D. J., Young, D. W., Church, N. J. & Spencer, P. in Bioluminescence and Chemiluminescence: Fundamental and Applied Aspects (eds Campbell, A. K., Krick, L. J. & Stanley, P. E.) 387–391 (John Wiley and Sons, Chichester, 1994)

    Google Scholar 

  12. Deluca, M. Firefly luciferase. Adv. Enzymol. 44, 37–68 (1976)

    CAS  PubMed  Google Scholar 

  13. Delarue, M. Aminoacyl-tRNA synthetases. Curr. Opin. Struct. Biol. 5, 48–55 (1995)

    Article  CAS  Google Scholar 

  14. Chang, K. H., Xiang, H. & Dunaway-Mariano, D. Acyl-adenylate motif of the acyl-adenylate/thioester-forming enzyme superfamily: a site-directed mutagenesis study with the Pseudomonas sp. strain CBS3 4-chlorobenzoate:coenzyme A ligase. Biochemistry 36, 15650–15659 (1997)

    Article  CAS  Google Scholar 

  15. Kleinkauf, H. & Von Dohren, H. A nonribosomal system of peptide biosynthesis. Eur. J. Biochem. 236, 335–351 (1996)

    Article  CAS  Google Scholar 

  16. Babbitt, P. C. et al. Ancestry of the 4-chlorobenzoate dehalogenase: analysis of amino acid sequence identities among families of acyl:adenyl ligases, enoyl-CoA hydratases/isomerases, and acyl-CoA thioesterases. Biochemistry 31, 5594–5604 (1992)

    Article  CAS  Google Scholar 

  17. May, J. J., Kessler, N., Marahiel, M. A. & Stubbs, M. T. Crystal structure of DhbE, an archetype for aryl acid activating domains of modular nonribosomal peptide synthetases. Proc. Natl Acad. Sci. USA 99, 12120–12125 (2002)

    Article  ADS  CAS  Google Scholar 

  18. Ueda, H. et al. X-ray crystallographic conformational study of 5′-O-[N-(l-alanyl)-sulfamoyl]adenosine, a substrate analogue for alanyl-tRNA synthetase. Biochim. Biophys. Acta 1080, 126–134 (1991)

    Article  CAS  Google Scholar 

  19. Branchini, B. R., Murtiashaw, M. H., Carmody, J. N., Mygatt, E. E. & Southworth, T. L. Synthesis of an N-acyl sulfamate analog of luciferyl-AMP: a stable and potent inhibitor of firefly luciferase. Bioorg. Med. Chem. Lett. 15, 3860–3864 (2005)

    Article  CAS  Google Scholar 

  20. Conti, E., Stachelhaus, T., Marahiel, M. A. & Brick, P. Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S. EMBO J. 16, 4174–4183 (1997)

    Article  CAS  Google Scholar 

  21. Kumasaka, T., Yamamoto, M., Yamashita, E., Moriyama, H. & Ueki, T. Trichromatic concept optimizes MAD experiments in synchrotron X-ray crystallography. Structure 10, 1205–1210 (2002)

    Article  CAS  Google Scholar 

  22. Adachi, S. et al. The RIKEN structural biology beamline II (BL44B2) at the SPring-8. Nucl. Instrum. Methods A 467–468, 711–714 (2001)

    Article  ADS  Google Scholar 

  23. Pflugrath, J. W. The finer things in X-ray diffraction data collection. Acta Crystallogr. D 55, 1718–1725 (1999)

    Article  CAS  Google Scholar 

  24. Leslie, A. G. W. in Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography No. 26 (CCP4, Warrington, 1992)

    Google Scholar 

  25. Navaza, J. AMoRe: an automated package for molecular replacement. Acta Crystallogr. A 50, 157–163 (1994)

    Article  Google Scholar 

  26. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    Article  CAS  Google Scholar 

  27. Perrakis, A., Morris, R. & Lamzin, V. S. Automated protein model building combined with iterative structure refinement. Nature Struct. Biol. 6, 458–463 (1999)

    Article  CAS  Google Scholar 

  28. Roussel, A. & Cambillau, C. Turbo-Frodo Manual (AFMB-CNRS, Marseille, 1996)

    Google Scholar 

  29. DeLano, W. L. The PyMOL Molecular Graphics System. (DeLano Scientific, San Carlos, California, 2002)

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We thank N. Kajiyama and K.Hirokawa (Kikkoman Corp.) for providing cDNA of luciferase and measurement of bioluminescence spectra. We also thank M. Yamamoto and S. Adachi (RIKEN Harima Institute) for X-ray diffraction data collection on the RIKEN beamlines BL45XU and BL44B2 at SPring-8. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (T.N. and H.K.)

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Correspondence to Hiroaki Kato.

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Atomic coordinates for luciferase structures have been deposited in the Protein Data Bank under accession codes 2D1Q (WT·Mg-ATP), 2D1S (WT·DLSA), 2D1R (WT·AMP/oxyluciferin) and 2D1T (S286N·DLSA). The authors declare no competing financial interests.

Supplementary information

Supplementary Notes

This file contains Supplementary Discussion, Supplementary Methods, Supplementary Figure Legends, Supplementary Movie Legends and Supplementary Table 1. (DOC 111 kb)

Supplementary Figures

This file contains Supplementary Figures 1–3. (PDF 1320 kb)

Supplementary Movie 1

A movie of the luminescence reaction of wild-type luciferase. (MOV 427 kb)

Supplementary Movie 2

A movie of the luminescence reaction of S286N mutant luciferase. (MOV 432 kb)

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Nakatsu, T., Ichiyama, S., Hiratake, J. et al. Structural basis for the spectral difference in luciferase bioluminescence. Nature 440, 372–376 (2006).

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