Unravelling the mechanism of pH-regulation in dinoflagellate luciferase (original) (raw)
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Engineering the protein dynamics of an ancestral luciferase
Nature Communications
Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable temp...
Physical Chemistry Chemical Physics, 2010
Firefly bioluminescence displays a sensitivity to pH changes through an alteration of the energy of the emitted photon leading to yellow-green light above BpH 6.5 and red light below this value. Calculations using the fragment molecular orbital method have been performed on the active site of the luciferase enzyme from the Japanese firefly Luciola cruciata in order to investigate both the importance of different protonation states and tautomeric forms of the lumophore, oxyluciferin, and the role played by protonation of the active site AMP molecule. The results suggest that whilst an equilibrium between several protonation/tautomeric states of oxyluciferin is possible, a single oxyluciferin species (the phenolate-keto form) may be mostly responsible for both emission colours, with changes in polarization by the active site caused by protonation of the AMP molecule playing an important role in mediating the pH-dependent shift.
Engineering Protein Dynamics of Ancestral Luciferase
2020
Insertion-deletion mutations are sources of major functional innovations in naturally evolved proteins, but directed evolution methods rely primarily on substitutions. Here, we report a powerful strategy for engineering backbone dynamics based on InDel mutagenesis of a stable and evolvable template, and its validation in application to a thermostable ancestor of haloalkane dehalogenase and Renilla luciferase. First, extensive multidisciplinary analysis linked the conformational flexibility of a loop-helix fragment to binding of the bulky substrate coelenterazine. The fragment’s key role in extant Renilla luciferase was confirmed by transplanting it into the ancestor. This increased its catalytic efficiency 7,000-fold, and fragment-containing mutants showed highly stable glow-type bioluminescence with 100-fold longer half-lives than the flash-type Renilla luciferase RLuc8, thereby addressing a limitation of a popular molecular probe. Thus, our three-step approach: (i) constructing a ...
Protein Science, 2001
Although the crystal structure of Vibrio harveyi luciferase has been elucidated, the binding sites for the flavin mononucleotide and fatty aldehyde substrates are still unknown. The determined location of the phosphate-binding site close to Arg 107 on the alpha subunit of luciferase is supported here by point mutagenesis. This information, together with previous structure-activity data for the length of the linker connecting the phosphate group to the isoalloxazine ring represent important characteristics of the luciferase-bound conformation of the flavin mononucleotide. A model of the luciferase-flavin complex is developed here using flexible docking supplemented by these structural constraints. The location of the phosphate moiety was used as the anchor in a flexible docking procedure performed by conformation search by using the Monte Carlo minimization approach. The resulting databases of energy-ranked feasible conformations of the luciferase complexes with flavin mononucleotide, omega-phosphopentylflavin, omega-phosphobutylflavin, and omega-phosphopropylflavin were filtered according to the structure-activity profile of these analogs. A unique model was sought not only on energetic criteria but also on the geometric requirement that the isoalloxazine ring of the active flavin analogs must assume a common orientation in the luciferase-binding site, an orientation that is also inaccessible to the inactive flavin analog. The resulting model of the bacterial luciferase-flavin mononucleotide complex is consistent with the experimental data available in the literature. Specifically, the isoalloxazine ring of the flavin mononucleotide interacts with the Ala 74-Ala 75 cis-peptide bond as well as with the Cys 106 side chain in the alpha subunit of luciferase. The model of the binary complex reveals a distinct cavity suitable for aldehyde binding adjacent to the isoalloxazine ring and flanked by other key residues (His 44 and Trp 250) implicated in the active site.
PloS one, 2017
Photoproteins are responsible for light emission in a variety of marine ctenophores and coelenterates. The mechanism of light emission in both families occurs via the same reaction. However, the arrangement of amino acid residues surrounding the chromophore, and the catalytic mechanism of light emission is unknown for the ctenophore photoproteins. In this study, we used quantum mechanics/molecular mechanics (QM/MM) and site-directed mutagenesis studies to investigate the details of the catalytic mechanism in berovin, a member of the ctenophore family. In the absence of a crystal structure of the berovin-substrate complex, molecular docking was used to determine the binding mode of the protonated (2-hydroperoxy) and deprotonated (2-peroxy anion) forms of the substrate to berovin. A total of 13 mutants predicted to surround the binding site were targeted by site-directed mutagenesis which revealed their relative importance in substrate binding and catalysis. Molecular dynamics simulat...
Enzyme and microbial technology, 2019
Firefly luciferase as a bioluminescent enzyme has many applications in various fields from scientific research to commercial goals. This enzyme is relatively unstable with low functional capacity due to rapid inactivation in physiological temperature, low in vitro stability and high susceptibility to proteolytic degradation. Based on previous studies, two regions 206-220 and 329-341 on N-domain of Photinus pyralis luciferase are known accessible and flexible. Flexible regions may lead to protein instability. Here, the effect of mutation at positively charged residues Lys(K)329 and Arg(R)330 on the stability of luciferase was studied. Furthermore, the role of these mutations on the structure and function was evaluated. Introducing of these point mutations did not affect the orientation of critical residues in bioluminescence color determination. The kinetic studies showed that thermostability and Km value for luciferin in both mutants were decreased as compared to wild type. However, optimum pH and optimum temperature showed no significant changes in both mutants. Moreover, the structural data revealed an increase in tryptophan fluorescence intensity and secondary structure content for R330Q in compared with wild type, while intrinsic fluorescence and far-UV CD intensity in K329I mutant was decreased.
Proceedings of the National Academy of Sciences, 2004
Enzymes with multiple catalytic sites are rare, and their evolutionary significance remains to be established. This study of luciferases from seven dinoflagellate species examines the previously undescribed evolution of such proteins. All these enzymes have the same unique structure: three homologous domains, each with catalytic activity, preceded by an N-terminal region of unknown function. Both pairwise comparison and phylogenetic inference indicate that the similarity of the corresponding individual domains between species is greater than that between the three different domains of each polypeptide. Trees constructed from each of the three individual domains are congruent with the tree of the full-length coding sequence. Luciferase and ribosomal DNA trees both indicate that the Lingulodinium polyedrum luciferase diverged early from the other six. In all species, the amino acid sequence in the central regions of the three domains is strongly conserved, suggesting it as the catalyt...
Bioluminescent and structural features of native folded Gaussia luciferase
Journal of Photochemistry and Photobiology B: Biology, 2018
The secreted luciferases responsible for light emission of marine copepods have gained popularity for being used in noninvasive imaging of intracellular events. The secreted luciferase of copepod Gaussia princeps is a one-subunit protein catalyzing coelenterazine oxidation to emit blue light. It consists of the N-terminal variable part that bears a signal peptide for secretion and the C-terminal catalytic domain containing ten highly conserved Cys residues supposing the existence of up to five S-S bonds. Despite wide application of Gaussia luciferase in biomedical research, its biochemical properties are still insufficiently studied due to the general problem of obtaining the proper folded Cys-rich proteins in bacterial cells. Here we report the properties of the proper folded Gaussia luciferase produced in insect cells using baculovirus expression system. This high purity luciferase reveals the highest activity at 15-20 o C but retains only ~20% activity at 37 o C that may hamper its application for in vivo assays. The maximum of bioluminescent activity of GpLuc is found at NaCl concentrations in the range of 1.0-1.5 M and, furthermore, a high NaCl concentration enhances luciferase stability to thermal denaturation, i.e. Gaussia luciferase displays the features characteristic of halophilic enzymes. The studies on bioluminescence kinetics at different coelenterazine concentrations obviously show a positive cooperativity of Gaussia luciferase with coelenterazine (Hill coefficient-1.8 ± 0.2; K 0.5-2.14 ± 0.17 µM). We suggest this effect to be rather due to the so-called kinetic cooperativity conditioned by conformational changes in response to substrate binding than to the presence of two catalytic sites.