Crystal Structures of the Luciferase and Green Fluorescent Protein from Renilla reniformis

Molecular Imaging Program at Stanford, Department of Radiology and Bio-X Program, The James H. Clark Center, Stanford University School of Medicine, 318 Campus Drive, Clark E150, Stanford, CA 94305-5427, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 01/2008; 374(4):1017-28. DOI: 10.1016/j.jmb.2007.09.078
Source: PubMed


Due to its ability to emit light, the luciferase from Renilla reniformis (RLuc) is widely employed in molecular biology as a reporter gene in cell culture experiments and small animal imaging. To accomplish this bioluminescence, the 37-kDa enzyme catalyzes the degradation of its substrate coelenterazine in the presence of molecular oxygen, resulting in the product coelenteramide, carbon dioxide, and the desired photon of light. We successfully crystallized a stabilized variant of this important protein (RLuc8) and herein present the first structures for any coelenterazine-using luciferase. These structures are based on high-resolution data measured to 1.4 A and demonstrate a classic alpha/beta-hydrolase fold. We also present data of a coelenteramide-bound luciferase and reason that this structure represents a secondary conformational form following shift of the product out of the primary active site. During the course of this work, the structure of the luciferase's accessory green fluorescent protein (RrGFP) was also determined and shown to be highly similar to that of Aequorea victoria GFP.

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Available from: Andreas Markus Loening, Jan 06, 2016
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    • "Bioluminescence is a phenomenon that is generally produced by a luciferin-luciferase reaction with molecular oxygen and has been reported from bacteria to insects[1,2]. The crystal structure analyses of luciferases have been performed, and the structures of bacterial luciferase (a heterodimer of a-subunit (40 kDa) and bsubunits (37 kDa), PDB ID: 1LUC)[3], firefly luciferase (62 kDa, PDB ID: 1LCI)[4], a mutated Renilla luciferase (34 kDa, PDB ID: 2PSJ)[5], and the 42 kDa catalytic domain (D3) of dinoflagellate luciferase (PDB ID: 1VPR)[6]have been determined. These luciferases oxidize specific luciferins with molecular oxygen in the absence or presence of co-factors, and produce oxyluciferin with emission of light. "
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    ABSTRACT: The 19 kDa protein (KAZ) of Oplophorus luciferase is a catalytic component, that oxidizes coelenterazine (a luciferin) with molecular oxygen to emit light. The crystal structure of the mutated 19 kDa protein (nanoKAZ) was determined at 1.71 Å resolution. The structure consists of 11 antiparallel β-strands forming a β-barrel that is capped by 4 short α-helices. The structure of nanoKAZ is similar to those of fatty acid-binding proteins (FABPs), even though the amino acid sequence similarity was very low between them. The coelenterazine-binding site and the catalytic site for the luminescence reaction might be in a central cavity of the β-barrel structure.
    Full-text · Article · Dec 2015 · Biochemical and Biophysical Research Communications
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    • "From the available crystal structures of cofactorless oxygenases (Grocholski et al., 2010; Loening et al., 2007; Sciara et al., 2003; Steiner et al., 2010; Widboom et al., 2007) and oxidases (Carlson et al., 2008; Colloc'h et al., 1997, 2008; Juan et al., 2008; Lee et al., 2005; Schwarzenbacher et al., 2004), it has become evident that different protein folds can provide the catalytic scaffold for cofactor-independent O 2 activation. Nevertheless, the enzymes appear to share common mechanistic features. "
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    ABSTRACT: In contrast to the majority of O2-activating enzymes, which depend on an organic cofactor or a metal ion for catalysis, a particular group of structurally unrelated oxygenases is functional without any cofactor. In this study, we characterized the mechanism of O2 activation in the reaction pathway of a cofactor-independent dioxygenase with an α/β-hydrolase fold, which catalyzes the oxygenolytic cleavage of 2-alkyl-3-hydroxy-4(1H)-quinolones. Chemical analysis and electron paramagnetic resonance spectroscopic data revealed that O2 activation in the enzyme's active site is substrate-assisted, relying on single electron transfer from the bound substrate anion to O2 to form a radical pair, which recombines to a C2-peroxide intermediate. Thus, an oxygenase can function without a cofactor, if the organic substrate itself, after activation to a (carb)anion by an active-site base, is intrinsically reactive toward molecular oxygen.
    Preview · Article · Dec 2013 · Chemistry & biology
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    • "The three dimensional structure of RLuc has been solved. It is a 37 kDa monomer and it contains a single substrate binding site [9]. CLuc is a 62 kDa protein [10]. "
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    ABSTRACT: Marine luciferases are increasingly used as reporters to study gene regulation. These luciferases have utility in bioluminescent assay development, although little has been reported on their catalytic properties in response to substrate concentration. Here, we report that the two marine luciferases from the copepods, Gaussia princeps (GLuc) and Metridia longa (MLuc) were found, surprisingly, to produce light in a cooperative manner with respect to their luciferin substrate concentration; as the substrate concentration was decreased 10 fold the rate of light production decreased 1000 fold. This positive cooperative effect is likely a result of allostery between the two proposed catalytic domains found in Gaussia and Metridia. In contrast, the marine luciferases from Renilla reniformis (RLuc) and Cypridina noctiluca (CLuc) demonstrate a linear relationship between the concentration of their respective luciferin and the rate of light produced. The consequences of these enzyme responses are discussed.
    Full-text · Article · Jun 2012 · PLoS ONE
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