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Eugene S. Vysotski - One of the best experts on this subject based on the ideXlab platform.
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Unusual shift in the visible absorption spectrum of an active ctenophore photoprotein elucidated by time-dependent density functional theory
Photochemical & Photobiological Sciences, 2021Co-Authors: Felix N. Tomilin, Ludmila P Burakova, Anastasia V. Rogova, Olga N. Tchaikovskaya, Pavel V. Avramov, Dmitri G. Fedorov, Eugene S. VysotskiAbstract:Active hydromedusan and ctenophore Ca^2+-regulated photoproteins form complexes consisting of apoprotein and strongly non-covalently bound 2-hydroperoxyCoelenterazine (an oxygenated intermediate of Coelenterazine). Whereas the absorption maximum of hydromedusan photoproteins is at 460–470 nm, ctenophore photoproteins absorb at 437 nm. Finding out a physical reason for this blue shift is the main objective of this work, and, to achieve it, the whole structure of the protein–substrate complex was optimized using a linear scaling quantum–mechanical method. Electronic excitations pertinent to the spectra of the 2-hydroperoxy adduct of Coelenterazine were simulated with time-dependent density functional theory. The dihedral angle of 60° of the 6-( p -hydroxy)-phenyl group relative to the imidazopyrazinone core of 2-hydroperoxyCoelenterazine molecule was found to be the key factor determining the absorption of ctenophore photoproteins at 437 nm. The residues relevant to binding of the substrate and its adopting the particular rotation were also identified.
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bioluminescent properties of semi synthetic obelin and aequorin activated by Coelenterazine analogues with modifications of c 2 c 6 and c 8 substituents
International Journal of Molecular Sciences, 2020Co-Authors: Elena V. Eremeeva, Natalia P Malikova, Tianyu Jiang, Eugene S. VysotskiAbstract:Ca2+-regulated photoproteins responsible for bioluminescence of a variety of marine organisms are single-chain globular proteins within the inner cavity of which the oxygenated Coelenterazine, 2-hydroperoxyCoelenterazine, is tightly bound. Alongside with native Coelenterazine, photoproteins can also use its synthetic analogues as substrates to produce flash-type bioluminescence. However, information on the effect of modifications of various groups of Coelenterazine and amino acid environment of the protein active site on the bioluminescent properties of the corresponding semi-synthetic photoproteins is fragmentary and often controversial. In this paper, we investigated the specific bioluminescence activity, light emission spectra, stopped-flow kinetics and sensitivity to calcium of the semi-synthetic aequorins and obelins activated by novel Coelenterazine analogues and the recently reported Coelenterazine derivatives. Several semi-synthetic photoproteins activated by the studied Coelenterazine analogues displayed sufficient bioluminescence activities accompanied by various changes in the spectral and kinetic properties as well as in calcium sensitivity. The poor activity of certain semi-synthetic photoproteins might be attributed to instability of some Coelenterazine analogues in solution and low efficiency of 2-hydroperoxy adduct formation. In most cases, semi-synthetic obelins and aequorins displayed different properties upon being activated by the same Coelenterazine analogue. The results indicated that the OH-group at the C-6 phenyl ring of Coelenterazine is important for the photoprotein bioluminescence and that the hydrogen-bond network around the substituent in position 6 of the imidazopyrazinone core could be the reason of different bioluminescence activities of aequorin and obelin with certain Coelenterazine analogues.
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Bioluminescent and structural features of native folded Gaussia luciferase.
'Elsevier BV', 2019Co-Authors: Marina D. Larionova, Svetlana V Markova, Eugene S. VysotskiAbstract: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 SS 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 °C but retains only ~20% activity at 37 °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; K0.5–2.14 ± 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
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Coelenterazine dependent luciferases
Biochemistry, 2015Co-Authors: Svetlana V Markova, Eugene S. VysotskiAbstract:Bioluminescence is a widespread natural phenomenon. Luminous organisms are found among bacteria, fungi, protozoa, coelenterates, worms, molluscs, insects, and fish. Studies on bioluminescent systems of various organisms have revealed an interesting feature - the mechanisms underlying visible light emission are considerably different in representatives of different taxa despite the same final result of this biochemical process. Among the several substrates of bioluminescent reactions identified in marine luminous organisms, the most commonly used are imidazopyrazinone derivatives such as Coelenterazine and Cypridina luciferin. Although the substrate used is the same, bioluminescent proteins that catalyze light emitting reactions in taxonomically remote luminous organisms do not show similarity either in amino acid sequences or in spatial structures. In this review, we consider luciferases of various luminous organisms that use Coelenterazine or Cypridina luciferin as a substrate, as well as modifications of these proteins that improve their physicochemical and bioluminescent properties and therefore their applicability in bioluminescence imaging in vivo.
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role of key residues of obelin in Coelenterazine binding and conversion into 2 hydroperoxy adduct
Journal of Photochemistry and Photobiology B-biology, 2013Co-Authors: Elena V. Eremeeva, Svetlana V Markova, Eugene S. Vysotski, Willem J H Van BerkelAbstract:Bioluminescence of a variety of marine organisms is caused by monomeric Ca(2+)-regulated photoproteins, to which a peroxy-substituted Coelenterazine, 2-hydroperoxyCoelenterazine, is firmly bound. From the spatial structure the side chains of Tyr138, His175, Trp179, and Tyr190 of obelin are situated within the substrate-binding pocket at hydrogen bond distances with different atoms of the 2-hydroperoxyCoelenterazine. Here we characterized several obelin mutants with substitutions of these residues regarding their bioluminescence, Coelenterazine binding, and kinetics of active obelin formation. We demonstrate that Tyr138, His175, Trp179, and Tyr190 are all important for Coelenterazine activation; substitution of any of these residues leads to significant decrease of the apparent reaction rate. The hydrogen bond network formed by Tyr138, Trp179 and Tyr190 participates in the proper positioning of Coelenterazine in the active site and subsequent stabilization of the 2-hydroperoxy adduct of Coelenterazine. His175 might serve as a proton shuttle during 2-hydroperoxyCoelenterazine formation.
Satoshi Inouye - One of the best experts on this subject based on the ideXlab platform.
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Chiral deaza-Coelenterazine analogs for probing a substrate-binding site in the Ca2+-binding photoprotein aequorin.
'Public Library of Science (PLoS)', 2021Co-Authors: Satoshi Inouye, Yuto Sumida, Yuri Tomabechi, Jumpei Taguchi, Mikako Shirouzu, Takamitsu HosoyaAbstract:The Ca2+-binding photoprotein aequorin is a complex of apoAequorin (apoprotein) and (S)-2-peroxyCoelenterazine. Aequorin can be regenerated by the incubation of apoAequorin with Coelenterazine and molecular oxygen (O2). In this study, to investigate the molecular recognition of apoAequorin for Coelenterazine using chemical probes, the chiral deaza-analogs of (S)- and (R)-deaza-CTZ (daCTZ) for Coelenterazine and of (S)-2- and (R)-2-hydroxymethyl-deaza-CTZ (HM-daCTZ) for 2-peroxyCoelenterazine were efficiently prepared by the improvement method. The chiral deaza-analogs of (S)-daCTZ and (S)-HM-daCTZ selectively inhibited the regeneration step to aequorin by binding the catalytic site of Coelenterazine in the apoAequorin molecule. The crystal structures of the apoAequorin complexes with (S)-daCTZ and (S)-HM-daCTZ were determined, suggesting that the hydroxy moiety at the C6-hydroxyphenyl group and the carbonyl moiety of the imidazopyrazinone ring in Coelenterazine are essential to bind the apoAequorin molecule through hydrogen bonding. Therefore, the chiral deaza-analogs of Coelenterazine can be used as a probe to study the interaction between Coelenterazine and the related proteins including photoprotein, luciferase, and Coelenterazine-binding protein
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crystal structure of nanokaz the mutated 19 kda component of oplophorus luciferase catalyzing the bioluminescent reaction with Coelenterazine
Biochemical and Biophysical Research Communications, 2016Co-Authors: Yuri Tomabechi, Takamitsu Hosoya, Mikako Shirouzu, Haruhiko Ehara, Shunichi Sekine, Satoshi InouyeAbstract: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 A 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.
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bioluminescence of the ca2 binding photoprotein aequorin after cysteine modification protein modification site specific mutagenesis protein active site oxygenase protein conformation
2016Co-Authors: Satoshi Inouye, Yoshiyuki Sakaki, Frederick I TsujitAbstract:Aequorin is a monomeric Ca2+-binding pro- tein (Mr, 21,400) that emits light upon reacting with Ca2+. The protein has three Ca2+-binding sites, three cysteine residues, and a noncovalently bound chromophore that consists of Coelenterazine and molecular oxygen. Light is emitted via an intramolecular reaction in which Coelenterazine is oxidized by the bound oxygen. After light emission, aequorin may be regenerated by incubating the protein with Coelenterazine, dissolved oxygen, EDTA, and 2-mercaptoethanol. To under- stand structure-function relationships in this protein, we used the technique of site-specific mutagenesis to replace the three cysteine residues with serine. Six of the seven modified aequor- ins had reduced luminescence activity, whereas the seventh with all three cysteines replaced by serine had luminescence activity equal to or greater than that of the wild-type aequorin. Further, the time required for the regeneration of the triply substituted aequorin was substantially increased compared to the time required for the regeneration of the wild-type ae- quorin. The results suggest that cysteine plays an important role in the regeneration of aequorin but not in its catalytic activity.
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a novel catalytic function of synthetic igg binding domain z domain from staphylococcal protein a light emission with Coelenterazine
Photochemistry and Photobiology, 2014Co-Authors: Satoshi Inouye, Yuiko SaharamiuraAbstract:The synthetic IgG-binding domain (Z domain) of staphylococcal protein A catalyzes the oxidation of Coelenterazine to emit light like a Coelenterazine-utilizing luciferase. The Z domain derivatives (ZZ-gCys, Z-gCys and Z-domain) were purified and the luminescence properties were characterized by comparing with Coelenterazine-utilizing luciferases, including Renilla luciferase, Gaussia luciferase and the catalytic 19 kDa protein of Oplophorus luciferase. Three Z domain derivatives showed luminescence activity with Coelenterazine and the order of the initial maximum intensity of luminescence was ZZ-gCys (100%) > Z-gCys (36.8%) > Z-domain (1.1%) > bovine serum albumin (BSA; 0.9%) > staphylococcal protein A (0.1%) and the background value of Coelenterazine (0.1%) in our conditions. The luminescence properties of ZZ-gCys showed the similarity to that of Gaussia luciferase, including the luminescence pattern, the emission spectrum, the stimulation by halogen ions and nonionic detergents and the substrate specificity for Coelenterazine analogues. In contrast, the luminescence properties of Z-gCys were close to the catalytic 19 kDa protein of Oplophorus luciferase. The catalytic region of the Z domain for the luminescence reaction might be different from the IgG-binding region of the Z domain.
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c6 deoxy Coelenterazine analogues as an efficient substrate for glow luminescence reaction of nanokaz the mutated catalytic 19 kda component of oplophorus luciferase
Biochemical and Biophysical Research Communications, 2013Co-Authors: Satoshi Inouye, Yuiko Saharamiura, Jun Ichi Sato, Suguru Yoshida, Hajime Kurakata, Takamitsu HosoyaAbstract:The codon-optimized gene for the mutated 19 kDa protein (nanoKAZ), which is the catalytic component of Oplophorus luciferase, was expressed in Escherichia coli cells and the recombinant protein was highly purified. The secretory expression of nanoKAZ from CHO-K1 cells was performed by fusing the secretory signal peptide sequence of Gaussia luciferase to the amino-terminus of nanoKAZ. The substrate specificity for the purified nanoKAZ and the nanoKAZ secreted into the cultured medium was determined, indicating that bis-Coelenterazine (bis-CTZ) and newly synthesized 6h-f-Coelenterazine (6h-f-CTZ) are an efficient substrate for the glow luminescence reaction of nanoKAZ.
Svetlana V Markova - One of the best experts on this subject based on the ideXlab platform.
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Bioluminescent and structural features of native folded Gaussia luciferase.
'Elsevier BV', 2019Co-Authors: Marina D. Larionova, Svetlana V Markova, Eugene S. VysotskiAbstract: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 SS 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 °C but retains only ~20% activity at 37 °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; K0.5–2.14 ± 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
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Coelenterazine dependent luciferases
Biochemistry, 2015Co-Authors: Svetlana V Markova, Eugene S. VysotskiAbstract:Bioluminescence is a widespread natural phenomenon. Luminous organisms are found among bacteria, fungi, protozoa, coelenterates, worms, molluscs, insects, and fish. Studies on bioluminescent systems of various organisms have revealed an interesting feature - the mechanisms underlying visible light emission are considerably different in representatives of different taxa despite the same final result of this biochemical process. Among the several substrates of bioluminescent reactions identified in marine luminous organisms, the most commonly used are imidazopyrazinone derivatives such as Coelenterazine and Cypridina luciferin. Although the substrate used is the same, bioluminescent proteins that catalyze light emitting reactions in taxonomically remote luminous organisms do not show similarity either in amino acid sequences or in spatial structures. In this review, we consider luciferases of various luminous organisms that use Coelenterazine or Cypridina luciferin as a substrate, as well as modifications of these proteins that improve their physicochemical and bioluminescent properties and therefore their applicability in bioluminescence imaging in vivo.
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role of key residues of obelin in Coelenterazine binding and conversion into 2 hydroperoxy adduct
Journal of Photochemistry and Photobiology B-biology, 2013Co-Authors: Elena V. Eremeeva, Svetlana V Markova, Eugene S. Vysotski, Willem J H Van BerkelAbstract:Bioluminescence of a variety of marine organisms is caused by monomeric Ca(2+)-regulated photoproteins, to which a peroxy-substituted Coelenterazine, 2-hydroperoxyCoelenterazine, is firmly bound. From the spatial structure the side chains of Tyr138, His175, Trp179, and Tyr190 of obelin are situated within the substrate-binding pocket at hydrogen bond distances with different atoms of the 2-hydroperoxyCoelenterazine. Here we characterized several obelin mutants with substitutions of these residues regarding their bioluminescence, Coelenterazine binding, and kinetics of active obelin formation. We demonstrate that Tyr138, His175, Trp179, and Tyr190 are all important for Coelenterazine activation; substitution of any of these residues leads to significant decrease of the apparent reaction rate. The hydrogen bond network formed by Tyr138, Trp179 and Tyr190 participates in the proper positioning of Coelenterazine in the active site and subsequent stabilization of the 2-hydroperoxy adduct of Coelenterazine. His175 might serve as a proton shuttle during 2-hydroperoxyCoelenterazine formation.
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Coelenterazine v ligated to ca2 triggered Coelenterazine binding protein is a stable and efficient substrate of the red shifted mutant of renilla muelleri luciferase
Analytical and Bioanalytical Chemistry, 2010Co-Authors: Galina A Stepanyuk, Svetlana V Markova, James Unch, Natalia P Malikova, John J Lee, Eugene S. VysotskiAbstract:It has been shown that the Coelenterazine analog, Coelenterazine-v, is an efficient substrate for a reaction catalyzed by Renilla luciferase. The resulting bioluminescence emission maximum is shifted to a longer wavelength up to 40 nm, which allows the use of some "yellow" Renilla luciferase mutants for in vivo imaging. However, the utility of Coelenterazine-v in small-animal imaging has been hampered by its instability in solution and in biological tissues. To overcome this drawback, we ligated Coelenterazine-v to Ca(2+)-triggered Coelenterazine-binding protein from Renilla muelleri, which apparently functions in the organism for stabilizing and protecting Coelenterazine from oxidation. The Coelenterazine-v bound within Coelenterazine-binding protein has revealed a greater long-term stability at both 4 and 37 °C. In addition, the Coelenterazine-binding protein ligated by Coelenterazine-v yields twice the total light over free Coelenterazine-v as a substrate for the red-shifted R. muelleri luciferase. These findings suggest the possibility for effective application of Coelenterazine-v in various in vitro assays.
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the intrinsic fluorescence of apo obelin and apo aequorin and use of its quenching to characterize Coelenterazine binding
FEBS Letters, 2009Co-Authors: Svetlana V Markova, Elena V. Eremeeva, Antonie J W G Visser, Willem J H Van Berkel, Adrie H Westphal, Eugene S. VysotskiAbstract:The intrinsic fluorescence of two apo-photoproteins has been characterized and its concentration-dependent quenching by Coelenterazine has been for the first time applied to determine the apparent dissociation constants for Coelenterazine binding with apo-aequorin (1.2 ± 0.12 μM) and apo-obelin (0.2 ± 0.04 μM). Stopped-flow measurements of fluorescence quenching showed that Coelenterazine binding is a millisecond-scale process, in contrast to the formation of an active photoprotein complex taking several hours. This finding evidently shows that the rate-limiting step of active photoprotein formation is the conversion of Coelenterazine into its 2-hydroperoxy derivative.
Ludmila A Frank - One of the best experts on this subject based on the ideXlab platform.
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recombinant metridia luciferase isoforms expression refolding and applicability for in vitro assay
Photochemical and Photobiological Sciences, 2008Co-Authors: Vasilisa V Borisova, Ludmila A Frank, Svetlana V Markova, Ludmila P Burakova, Eugene S. VysotskiAbstract:The recombinant Coelenterazine-dependent luciferases (isoforms MLuc164 and MLuc39) from the marine copepod Metridia longa were expressed as inclusion bodies in E. colicells, dissolved in 6 M guanidinium chloride and folded in conditions developed for proteins containing intramolecular disulfide bonds. One of them (MLuc39) was obtained in an active monomeric form with a high yield. The luciferase bioluminescence is found to be initiated not only by free Coelenterazine, but also by Ca2+-dependent Coelenterazine-binding protein (CBP) of Renilla muelleri on Ca2+ addition. The use of CBP as a “substrate” provides higher light emission and simultaneously the lower level of background. The high purity MLuc39 can be detected down to attomol with a linear range extending over 5 orders of magnitude. The MLuc39 reveals also a high stability towards heating and chemical modification; the chemically synthesized biotinylated derivatives of the luciferase preserve 35–40 % of the initial activity. The luciferase applicability as an in vitro bioluminescent reporter is demonstrated in model tandem bioluminescent solid-phase microassay combining the Ca2+-regulated photoprotein obelin and the Metridia luciferase.
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recombinant metridia luciferase isoforms expression refolding and applicability for in vitro assay
Photochemical and Photobiological Sciences, 2008Co-Authors: Vasilisa V Borisova, Ludmila A Frank, Svetlana V Markova, Ludmila P Burakova, Eugene S. VysotskiAbstract:The recombinant Coelenterazine-dependent luciferases (isoforms MLuc164 and MLuc39) from the marine copepod Metridia longa were expressed as inclusion bodies in E. colicells, dissolved in 6 M guanidinium chloride and folded in conditions developed for proteins containing intramolecular disulfide bonds. One of them (MLuc39) was obtained in an active monomeric form with a high yield. The luciferase bioluminescence is found to be initiated not only by free Coelenterazine, but also by Ca2+-dependent Coelenterazine-binding protein (CBP) of Renilla muelleri on Ca2+ addition. The use of CBP as a “substrate” provides higher light emission and simultaneously the lower level of background. The high purity MLuc39 can be detected down to attomol with a linear range extending over 5 orders of magnitude. The MLuc39 reveals also a high stability towards heating and chemical modification; the chemically synthesized biotinylated derivatives of the luciferase preserve 35–40 % of the initial activity. The luciferase applicability as an in vitro bioluminescent reporter is demonstrated in model tandem bioluminescent solid-phase microassay combining the Ca2+-regulated photoprotein obelin and the Metridia luciferase.
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Crystal structure of Coelenterazine-binding protein from Renilla muelleri at 1.7 Å: Why it is not a calcium-regulated photoprotein
Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology, 2008Co-Authors: Galina A Stepanyuk, Ludmila A Frank, Eugene S. Vysotski, Zhijie Liu, John Lee, Svetlana Markova, Bicheng WangAbstract:Bioluminescence in the sea pansy Renilla involves two distinct proteins, a Ca2+-triggered Coelenterazine-binding protein (CBP), and Renilla luciferase. CBP contains one tightly bound Coelenterazine molecule, which becomes available for reaction with luciferase and O2 only subsequent to Ca2+ binding. CBP belongs to the EF-hand superfamily of Ca2+-binding proteins and contains three “EF-hand” Ca2+-binding sites. The overall spatial structure of recombinant selenomethionine-labeled CBP determined at 1.7 A, is found to approximate the protein scaffold characteristic of the class of Ca2+-regulated photoproteins. Photoproteins however, catalyze molecular oxygen addition to Coelenterazine producing a 2-hydroperoxyCoelenterazine intermediate, which is stabilized within the binding cavity in the absence of Ca2+. Addition of Ca2+ triggers the bioluminescence reaction. However in CBP this first step of oxygen addition is not allowed. The different amino acid environments and hydrogen bond interactions within the binding cavity, are proposed to account for the different properties of the two classes of proteins.
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Coelenterazine binding protein of renilla muelleri cdna cloning overexpression and characterization as a substrate of luciferase
Photochemical and Photobiological Sciences, 2008Co-Authors: Maxim S Titushin, Ludmila A Frank, Svetlana V Markova, Eugene S. Vysotski, Galina A Stepanyuk, Natalia P Malikova, John J LeeAbstract:The Renilla bioluminescent system in vivo is comprised of three proteins—the luciferase, green-fluorescent protein, and Coelenterazine-binding protein (CBP), previously called luciferin-binding protein (LBP). This work reports the cloning of the full-size cDNA encoding CBP from soft coral Renilla muelleri, its overexpression and properties of the recombinant protein. The apo-CBP was quantitatively converted to CBP by simple incubation with Coelenterazine. The physicochemical properties of this recombinant CBP are determined to be practically the same as those reported for the CBP (LBP) of R. reniformis. CBP is a member of the four-EF-hand Ca2+-binding superfamily of proteins with only three of the EF-hand loops having the Ca2+-binding consensus sequences. There is weak sequence homology with the Ca2+-regulated photoproteins but only as a result of the necessary Ca2+-binding loop structure. In combination with Renilla luciferase, addition of only one Ca2+ is sufficient to release the Coelenterazine as a substrate for the luciferase for bioluminescence. This combination of the two proteins generates bioluminescence with higher reaction efficiency than using free Coelenterazine alone as the substrate for luciferase. This increased quantum yield, a difference of bioluminescence spectra, and markedly different kinetics, implicate that a CBP-luciferase complex might be involved.
Yuichi Oba - One of the best experts on this subject based on the ideXlab platform.
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etmopterus lantern sharks use Coelenterazine as the substrate for their luciferin luciferase bioluminescence system
Biochemical and Biophysical Research Communications, 2021Co-Authors: Gaku Mizuno, Daichi Yano, Jose Paitio, Hiromitsu Endo, Yuichi ObaAbstract:Abstract The lantern shark genus Etmopterus contains approximately 40 species of deep-sea bioluminescent cartilaginous fishes. They emit blue light mainly from the ventral body surface. The biological functions of this bioluminescence have been discussed based on the luminescence patterns, but the bioluminescence mechanism remains uncertain. In this study, we detected both Coelenterazine and Coelenterazine-dependent luciferase activity in the ventral photophore tissue of Etmopterus molleri. The results suggested that bioluminescence in lantern sharks is produced using Coelenterazine as the substrate for the luciferin-luciferase reaction, as some luminous bony fishes.
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etmopterus lantern sharks use Coelenterazine as the substrate for their luciferin luciferase bioluminescence system
bioRxiv, 2021Co-Authors: Gaku Mizuno, Daichi Yano, Jose Paitio, Hiromitsu Endo, Yuichi ObaAbstract:The lantern shark genus Etmopterus is a group of deep-sea bioluminescent fishes. They emit blue light mainly from the ventral body surface, and the primary biological function is considered to be for camouflage by counterillumination. In this study, we detected both Coelenterazine and Coelenterazine-specific luciferase activity in the ventral photophore tissues. The results suggested that bioluminescence in lantern sharks is produced using Coelenterazine as the substrate for the luciferin-luciferase reaction.
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lantern shark etmopterus use Coelenterazine as substrate for their luciferin luciferase bioluminescence system
bioRxiv, 2021Co-Authors: Gaku Mizuno, Daichi Yano, Jose Paitio, Hiromitsu Endo, Yuichi ObaAbstract:The lantern shark genus Etmopterus is a group of deep-sea bioluminescent fishes. They emit blue light mainly from the ventral body surface, and the primary biological function is considered to be for camouflage by counterillumination. In this study, we detected both Coelenterazine and Coelenterazine-specific luciferase activity in the ventral photophore tissues. The results suggested that bioluminescence in lantern sharks is produced using Coelenterazine as the substrate for the luciferin-luciferase reaction.
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Biosynthesis of Coelenterazine in the deep-sea copepod, Metridia pacifica.
Biochemical and biophysical research communications, 2009Co-Authors: Yuichi Oba, Shin-ichi Kato, Makoto Ojika, Satoshi InouyeAbstract:Coelenterazine is an imidazopyrazinone compound (3,7-dihydroimidazopyrazin-3-one structure) that is widely distributed in marine organisms and used as a luciferin for various bioluminescence reactions. We have used electrospray ionization-ion trap-mass spectrometry to investigate whether the deep-sea luminous copepod Metridia pacifica is able to synthesize Coelenterazine. By feeding experiments using deuterium labeled amino acids of l-tyrosine and l-phenylalanine, we have shown that Coelenterazine can be synthesized from two molecules of l-tyrosine and one molecule of l-phenylalanine in M. pacifica. This is the first demonstration that Coelenterazine is biosynthesized from free l-amino acids in a marine organism.
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identification of the luciferin luciferase system and quantification of Coelenterazine by mass spectrometry in the deep sea luminous ostracod conchoecia pseudodiscophora
ChemBioChem, 2004Co-Authors: Yuichi Oba, Shin-ichi Kato, Makoto Ojika, Hiroshi Tsuduki, Satoshi InouyeAbstract:The bioluminescence system of the ostracod Conchoecia pseudodiscophora, which is abundant in the Sea of Japan, has been characterized. The luminescence (lambda(max)=463 nm) is produced by a luciferin-luciferase reaction, and the luciferin has been identified as Coelenterazine. Coelenterazine, coelenteramide, and coelenteramine from C. pseudodiscophora were quantified by LC-ESI-MS/MS analysis. The Coelenterazine content was estimated to be approximately 230 pg per animal by using a calibration curve of synthetic Coelenterazine. The reaction between homogenates of C. pseudodiscophora and synthetic Coelenterazine showed luminescence activity; this suggests that a Coelenterazine-type luciferase is present.