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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    • "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.
    Chemistry & biology 12/2013; 21(2). DOI:10.1016/j.chembiol.2013.11.013 · 6.65 Impact Factor
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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.
    PLoS ONE 06/2012; 7(6):e40099. DOI:10.1371/journal.pone.0040099 · 3.23 Impact Factor
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    • "Several oxidases and oxygenases have been identified that require neither a metal ion nor an organic cofactor for activity, which raises the question of how these enzymes work. From the available crystal structures, it has become evident that different families of cofactor-independent oxygenases and oxidases exist, which do not share common structural motifs, i.e., different protein folds can provide the scaffold for cofactor-independent oxygenation and dioxygen reduction (Colloc'h et al. 1997; Dierks et al. 2005; Juan et al. 2008; Lee et al. 2005; Loening et al. 2007; Phillips et al. 2004; Schwarzenbacher et al. 2004; Sciara et al. 2003; Steiner et al. 2010; Widboom et al. 2007). This review updates the topic of cofactor-independent oxygenases addressed earlier (Fetzner 2002), includes an overview on mechanistically related oxidases and hypothesizes on the possibility of a common catalytic concept. "
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    ABSTRACT: Whereas the majority of O(2)-metabolizing enzymes depend on transition metal ions or organic cofactors for catalysis, a significant number of oxygenases and oxidases neither contain nor require any cofactor. Among the cofactor-independent oxidases, urate oxidase, coproporphyrinogen oxidase, and formylglycine-generating enzyme are of mechanistic as well as medical interest. Formylglycine-generating enzyme is also a promising tool for protein engineering as it can be used to equip proteins with a reactive aldehyde function. PqqC, an oxidase in the biosynthesis of the bacterial cofactor pyrroloquinoline quinone, catalyzes an eight-electron ring-closure oxidation reaction. Among bacterial oxygenases, quinone-forming monooxygenases involved in the tailoring of polyketides, the dioxygenase DpgC found in the biosynthesis of a building block of vancomycin and teicoplanin antibiotics, luciferase monooxygenase from Renilla sp., and bacterial ring-cleaving 2,4-dioxygenases active towards 3-hydroxy-4(1H)-quinolones have been identified as cofactor-independent enzymes. Interestingly, the 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases as well as Renilla luciferase use an alpha/beta-hydrolase architecture for oxygenation reactions. Cofactor-independent oxygenases and oxidases catalyze very different reactions and belong to several different protein families, reflecting their diverse origin. Nevertheless, they all may share the common mechanistic concept of initial base-catalyzed activation of their organic substrate and "substrate-assisted catalysis".
    Applied Microbiology and Biotechnology 02/2010; 86(3):791-804. DOI:10.1007/s00253-010-2455-0 · 3.34 Impact Factor
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