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Review
. 2010 Feb;67(3):387-404.
doi: 10.1007/s00018-009-0170-8. Epub 2009 Oct 27.

Firefly luciferase: an adenylate-forming enzyme for multicatalytic functions

Satoshi Inouye  1
Affiliations
Review

Firefly luciferase: an adenylate-forming enzyme for multicatalytic functions

Satoshi Inouye. Cell Mol Life Sci. 2010 Feb.

Abstract

Firefly luciferase is a member of the acyl-adenylate/thioester-forming superfamily of enzymes and catalyzes the oxidation of firefly luciferin with molecular oxygen to emit light. Knowledge of the luminescence mechanism catalyzed by firefly luciferase has been gathered, leading to the discovery of a novel catalytic function of luciferase. Recently, we demonstrated that firefly luciferase has a catalytic function of fatty acyl-CoA synthesis from fatty acids in the presence of ATP, Mg(2+) and coenzyme A. Based on identification of fatty acyl-CoA genes in firefly, Drosophila, and non-luminous click beetles, we then proposed that the evolutionary origin of firefly luciferase is a fatty acyl-CoA synthetase in insects. Further, we succeeded in converting the fatty acyl-CoA synthetase of non-luminous insects into functional luciferase showing luminescence activity by site-directed mutagenesis.

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Figures

Fig. 1
Fig. 1
Chemical structures of ATP, AMP and coenzyme A (CoA)
Fig. 2
Fig. 2
Chemical structures of firefly luciferin and its related compounds
Fig. 3
Fig. 3
Mechanism of the bioluminescence reaction of firefly luciferin by firefly luciferase
Fig. 4
Fig. 4
Proposed mechanisms for light emission in the firefly luciferase reaction
Fig. 5
Fig. 5
Luminescence pattern of firefly luciferase in the presence and absence of coenzyme A. The reaction mixture (100 μl) contained firefly luciferin (10 μM), ATP (250 μM) and MgCl2 (5 mM) in 100 mM Tris–HCl (pH 7.8). In the presence and absence of CoA (250 μM), the reaction was started by the addition of recombinant P. pyralis luciferase (1 pmol: Promega) and the light intensity of luminescence was measured at 22–23°C
Fig. 6
Fig. 6
Formation of dehydroluciferin, dehydroluciferyl CoA and d-luciferyl CoA from d- and l-luciferin during the luminescence reaction by firefly luciferase
Fig. 7
Fig. 7
Biosynthesis of firefly luciferin and conversion to firefly luciferin form oxyluciferin
Fig. 8
Fig. 8
Firefly luciferin analogues for the luciferase reaction and its luminescence activity. Plus and minus in parentheses show the positive and negative activities of luminescence as a substrate
Fig. 9
Fig. 9
Luciferin derivatives for bioluminescence assays using firefly luciferase
Fig. 10
Fig. 10
Inhibitors of the firefly luciferase–luciferin reaction
Fig. 11
Fig. 11
Comparison of chemical structures between luciferyl adenylate and its analogue DLSA
Fig. 12
Fig. 12
Structure of the firefly L. cruciata luciferase–DLSA complex. a Ribbon diagram of firefly luciferase in complex with DLSA (red). The N-terminal large and C-terminal small domains are drawn in gray and green, respectively, b A schematic representation of the DLSA binding area in firefly luciferase. The possible hydrogen bonds are shown by a green dashed line, and light blue circles show water molecules. The amino acid residues labeled with red and black show the hydrophobic contacts with DLSA
Fig. 13
Fig. 13
Determination of fatty acyl-CoA synthesis through fatty acyl adenylate from fatty acid, catalyzed by firefly luciferase. Asterisks indicate 14C-labeled position
Fig. 14
Fig. 14
A phylogenetic reconstruction of luciferases and their similar genes in insects. Numbers on the nodes indicate bootstrap values % from 10,000 replicates, and only the values over 50% are shown. Horizontal branch lengths indicate the genetic distances. GenBank accession number (Protein/Nucleotide) is shown in parentheses
Fig. 15
Fig. 15
Comparison of the amino acid sequences of AbLL (AbLL, AB353886), P. plagiophthalmus luciferase (PplLuc; AF543373), P. pyralis luciferase (PpyLuc; M15077) and L. cruciata luciferase (LcuLuc; M26194). Letters in the black boxes and shaded boxes indicate the identical amino acids and the similar amino acid groups with AbLL, respectively. The groups are defined as follows: A, S, T, P, and G; N, D, E, and Q; H, R, and K; M, L, I, and V; F, Y, and W. Gaps were inserted to assist in sequence alignment. The numbers on the right margin indicate the position of amino acid residues. Double arrow indicates the AMP-binding motif. Three asterisks indicate the positions of mutation in AbLL. The linker region between the N-terminal domain and C-terminal domain is shown by the sharp symbols
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