Firefly Luciferase Produces Hydrogen Peroxide as a Coproduct in Dehydroluciferyl Adenylate Formation

Fraga, Hugo; Fernandes, Diogo; Novotný, Jiřı́; Fontes, Rui; Silva, Joaquim C. G. Esteves da

doi:10.1002/cbic.200500443

Cited by 54 publications

(43 citation statements)

References 43 publications

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“…The smaller fragment sizes for the split RL strategy (23 kDa for NRL and 13 kDa for CRL) relative to split FL fragments (f32 kDa) may minimize steric hindrance for Hsp90a/ p23 and Hsp90h/p23 interactions in the split reporters. Because FL requires ATP for its enzymatic activity (43), SFL-PFAC may not be suitable for evaluating the efficacy of Hsp90 inhibitors (and other Feasibility of using SRL-PFAC for development of isoformselective Hsp90 inhibitors. Given (a) the Hsp90 chaperone system is highly up-regulated in multiple cancers (37), (b) both Hsp90a and Hsp90h are expressed in cancer cells and may play distinct roles in determining drug responses, and (c) the expression of Hsp90a and Hsp90h can be compensatory (38), Hsp90 inhibitors aimed at disruption of Hsp90a/p23 and Hsp90h/p23 interactions specifically or both interactions are being developed through indirect imaging of Hsp90a/p23 and Hsp90h/p23 interactions in conjunction with high-throughput screening.…”

Section: Discussionmentioning

confidence: 99%

Molecular Imaging of the Efficacy of Heat Shock Protein 90 Inhibitors in Living Subjects

et al. 2008

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Heat shock protein 90A (Hsp90A)/p23 and Hsp90B/p23 interactions are crucial for proper folding of proteins involved in cancer and neurodegenerative diseases. Small molecule Hsp90 inhibitors block Hsp90A/p23 and Hsp90B/p23 interactions in part by preventing ATP binding to Hsp90. The importance of isoform-selective Hsp90A/p23 and Hsp90B/p23 interactions in determining the sensitivity to Hsp90 was examined using 293T human kidney cancer cells stably expressing split Renilla luciferase (RL) reporters. Interactions between Hsp90A/p23 and Hsp90B/p23 in the split RL reporters led to complementation of RL activity, which was determined by bioluminescence imaging of intact cells in cell culture and living mice using a cooled charge-coupled device camera. The three geldanamycin-based and seven purinescaffold Hsp90 inhibitors led to different levels of inhibition of complemented RL activities (10-70%). However, there was no isoform selectivity to both classes of Hsp90 inhibitors in cell culture conditions. The most potent Hsp90 inhibitor, PU-H71, however, led to a 60% and 30% decrease in RL activity (14 hr) in 293T xenografts expressing Hsp90A/p23 and Hsp90B/p23 split reporters respectively, relative to carrier control-treated mice. Molecular imaging of isoform-specific Hsp90A/p23 and Hsp90B/p23 interactions and efficacy of different classes of Hsp90 inhibitors in living subjects have been achieved with a novel genetically encoded reporter gene strategy that should help in accelerating development of potent and isoformselective Hsp90 inhibitors. [Cancer Res 2008;68(1):216-26]

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Section: Discussionmentioning

confidence: 99%

Molecular Imaging of the Efficacy of Heat Shock Protein 90 Inhibitors in Living Subjects

et al. 2008

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“…This effect could be exerted not directly by L but instead through its adenylylation to L-AMP. L-AMP is a very strong luciferase inhibitor (20,23,39,42,43) and its synthesis from LH 2 -AMP can account for about 20% of the LH 2 consumed (40,41).…”

Section: Firefly Luciferase Reactionsmentioning

confidence: 99%

Firefly bioluminescence: A mechanistic approach of luciferase catalyzed reactions

2008

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SummaryLuciferase is a general term for enzymes catalyzing visible light emission by living organisms (bioluminescence). The studies carried out with Photinus pyralis (firefly) luciferase allowed the discovery of the reaction leading to light production. It can be regarded as a two-step process: the first corresponds to the reaction of luciferase's substrate, luciferin (LH 2 ), with ATPMg 21 generating inorganic pyrophosphate and an intermediate luciferyl-adenylate (LH 2 -AMP); the second is the oxidation and decarboxylation of LH 2 -AMP to oxyluciferin, the light emitter, producing CO 2 , AMP, and photons of yellow-green light (550-570 nm). In a dark reaction LH 2 -AMP is oxidized to dehydroluciferyl-adenylate (L-AMP). Luciferase also shows acyl-coenzyme A synthetase activity, which leads to the formation of dehydroluciferyl-coenzyme A (L-CoA), luciferyl-coenzyme A (LH 2 -CoA), and fatty acyl-CoAs. Moreover luciferase catalyzes the synthesis of dinucleoside polyphosphates from nucleosides with at least a 3 0 -phosphate chain plus an intact terminal pyrophosphate moiety. The LH 2 stereospecificity is a particular feature of the bioluminescent reaction where each isomer, D-LH 2 or L-LH 2 , has a specific function. Practical applications of the luciferase system, either in its native form or with engineered proteins, encloses the analytical assay of metabolites like ATP and molecular biology studies with luc as a reporter gene, including the most recent and increasing field of bioimaging.2008 IUBMB IUBMB Life, 61(1):

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“…[3][4][5][6] The chemical reaction that transforms LH 2 into oxyluciferin (OxyLH 2 ) is clearly defined. [6][7][8][9] However, many details of the chemical origin of the multicolor bioluminescence are ambiguous and challenge both theory and experiment. Experimental studies of the light emitters are difficult due to the great instability of OxyLH 2 in the excited state.…”

Section: Introductionmentioning

confidence: 99%

A Time‐Dependent Density Functional Theory Investigation on the Origin of Red Chemiluminescence

et al. 2010

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Is the resonance-based anionic keto form of oxyluciferin the chemical origin of multicolor bioluminescence? Can it modulate green into red luminescence? There is as yet no definitive answer from experiment or theory. The resonance-based anionic keto forms of oxyluciferin have been proposed as a cause of multicolor bioluminescence in the firefly. We model the possible structures by adding sodium or ammonium cations and investigating the ground- and excited-state geometries as well as the electronic absorption and emission spectra. A role for the resonance structures is obvious in the gas phase. The absorption and emission spectra of the two structures are quite different--one in the blue and another in the red. The differences in the spectra of the models are small in aqueous solution, with all the absorption and emission spectra in the yellow-green region. The resonance-based anionic keto form of oxyluciferin may be one origin of the red-shifted luminescence but is not the exclusive explanation for the variation from green (approximately 530 nm) to red (approximately 635 nm). We study the geometries, absorption, and emission spectra of the possible protonated compounds of keto(-1) in the excited states. A new emitter keto(-1)'-H is considered.

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Firefly Luciferase Produces Hydrogen Peroxide as a Coproduct in Dehydroluciferyl Adenylate Formation

Cited by 54 publications

References 43 publications

Molecular Imaging of the Efficacy of Heat Shock Protein 90 Inhibitors in Living Subjects

Molecular Imaging of the Efficacy of Heat Shock Protein 90 Inhibitors in Living Subjects

Firefly bioluminescence: A mechanistic approach of luciferase catalyzed reactions

A Time‐Dependent Density Functional Theory Investigation on the Origin of Red Chemiluminescence

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