Letter | Published:

Structural basis for the spectral difference in luciferase bioluminescence

Nature volume 440, pages 372376 (16 March 2006) | Download Citation



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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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.)

Author information


  1. Kinetic Crystallography Research Team, Membrane Dynamics Research Group, RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan

    • Toru Nakatsu
    • , Susumu Ichiyama
    • , Nobuyuki Kobashi
    •  & Hiroaki Kato
  2. Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan

    • Toru Nakatsu
    •  & Hiroaki Kato
  3. Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan

    • Jun Hiratake
    • , Adrian Saldanha
    •  & Kanzo Sakata


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Competing interests

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.

Corresponding author

Correspondence to Hiroaki Kato.

Supplementary information

Word documents

  1. 1.

    Supplementary Notes

    This file contains Supplementary Discussion, Supplementary Methods, Supplementary Figure Legends, Supplementary Movie Legends and Supplementary Table 1.

PDF files

  1. 1.

    Supplementary Figures

    This file contains Supplementary Figures 1–3.


  1. 1.

    Supplementary Movie 1

    A movie of the luminescence reaction of wild-type luciferase.

  2. 2.

    Supplementary Movie 2

    A movie of the luminescence reaction of S286N mutant luciferase.

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