Coelenterazine-dependent luciferases

Luciferase of copepod Metridia longa (MLuc) is a naturally secreted enzyme catalyzing the oxidative decarboxylation of coelenterazine with the emission of light. To date, three nonallelic isoforms of different lengths (17-24 kDa) for M. longa luciferase have been cloned. All the isoforms are single-chain proteins consisting of a 17-residue signal peptide for secretion, variable N-terminal part and conservative C-terminus responsible for luciferase activity. In contrast to other bioluminescent proteins containing a lot of aromatic residues which are frequently involved in light emission reaction, the C-terminal part of MLuc contains only four Phe, two Tyr, one Trp and two His residues. To figure out whether Tyr residues influence bioluminescence, we constructed the mutants with substitution of Tyr to Phe (Y72F and Y80F). Tyrosine substitutions do not eliminate the ability of luciferase to bioluminescence albeit significantly reduce relative specific activity and change bioluminescence kinetics. In addition, the Tyr replacements have no effect on bioluminescence spectrum, thereby indicating that tyrosines are not involved in the emitter formation. However, as it was found that the intrinsic fluorescence caused by Tyr residues is quenched by a reaction substrate, coelenterazine, in concentration-dependent manner, we infer that both tyrosine residues are located in the luciferase substrate-binding cavity.

Section: Resultsmentioning

confidence: 95%

Tyr72 and Tyr80 are Involved in the Formation of an Active Site of a Luciferase of Copepod Metridia longa

Larionova

Markova

Vysotski

2017

“…The identified bioluminescent enzymes from taxonomically unrelated organisms share no similarities in their amino acid sequences even when they use the same substrate for the bioluminescent reaction, as in the case of known coelenterazine‐dependent luciferases . Despite the wide variety of luminous organisms, the primary structures of just over ten types of luciferases with completely different sequences and properties were so far identified, supporting the idea of independent origin of these bioluminescent systems .…”

Section: Introductionmentioning

confidence: 82%

“…Like Renilla luciferase, the CopLucs catalyze a simple bioluminescent reaction of coelenterazine oxidation accompanied by blue light emission. But they do not share sequence or structural similarities with other cloned coelenterazine‐dependent luciferases of Renilla and Oplophorus families, and also not with the luciferases of Cypridina family which use the other imidazopyrazinone‐type luciferin as a substrate .…”

Section: Introductionmentioning

confidence: 89%

Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications

Markova

Larionova

Vysotski

2019

Copepod luciferases—a family of small secretory proteins of 18.4–24.3 kDa, including a signal peptide—are responsible for bright secreted bioluminescence of some marine copepods. The copepod luciferases use coelenterazine as a substrate to produce blue light in a simple oxidation reaction without any additional cofactors. They do not share sequence or structural similarity with other identified bioluminescent proteins including coelenterazine‐dependent Renilla and Oplophorus luciferases. The small size, strong luminescence activity and high stability, including thermostability, make secreted copepod luciferases very attractive candidates as reporter proteins which are particularly useful for nondisruptive reporter assays and for high‐throughput format. The most known and extensively investigated representatives of this family are the first cloned GpLuc and MLuc luciferases from copepods Gaussia princeps and Metridia longa, respectively. Immediately after cloning, these homologous luciferases were successfully applied as bioluminescent reporters in vivo and in vitro, and since then, the scope of their applications continues to grow. This review is an attempt to systemize and critically evaluate the data scattered through numerous articles regarding the main structural features of copepod luciferases, their luminescent and physicochemical properties. We also review the main trends of their application as bioluminescent reporters in cell and molecular biology.

“…3c), etc., is the same. The photoproteins can just be regarded as a luciferase having stabilized the reaction intermediate (104)(105)(106).…”

Section: Firefly and Coelenterazinementioning

confidence: 99%

Perspectives on Bioluminescence Mechanisms

Lee

2016

The molecular mechanisms of the bioluminescence systems of the firefly, bacteria and those utilizing imidazopyrazinone luciferins such as coelenterazine are gradually being uncovered using modern biophysical methods such as dynamic (ns-ps) fluorescence spectroscopy, NMR, X-ray crystallography and computational chemistry. The chemical structures of all reactants are well defined, and the spatial structures of the luciferases are providing important insight into interactions within the active cavity. It is generally accepted that the firefly and coelenterazine systems, although proceeding by different chemistries, both generate a dioxetanone high-energy species that undergoes decarboxylation to form directly the product in its S 1 state, the bioluminescence emitter. More work is still needed to establish the structure of the products completely. In spite of the bacterial system receiving the most research attention, the chemical pathway for excitation remains mysterious except that it is clearly not by a decarboxylation. Both the coelenterazine and bacterial systems have in common of being able to employ "antenna proteins," lumazine protein and the greenfluorescent protein, for tuning the color of the bioluminescence. Spatial structure information has been most valuable in informing the mechanism of the Ca 2+ -regulated photoproteins and the antenna protein interactions.