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

Abstract

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.

Bioluminescent and structural features of native folded Gaussia luciferase

“…respectively. The intensity of Tyr fluorescence in the case of GpLuc also exceeds that conditioned by Trp residue [11,34]. It obviously demonstrates that the environments of the side chains of aromatic residues in GpLuc and MLuc have to be identical and consequently their spatial structures should be very similar or the same.…”

“…luciferase isoforms [11,34], with excitation at 275 and 295 nm GpLuc fluoresces with the maxima at 303 and 330 nm characteristic of Tyr and Trp fluorescence (data not shown),…”

“…1). Recently it was demonstrated that Tyr residues may be involved in the formation of an active site of copepod luciferases [34]. Due to a low content of Tyr and Trp residues, the intrinsic fluorescence of copepod luciferases is rather weak.…”

“…With regard to the predicted secondary structure (Fig. S1), GpLuc is poor in -helices and -strands but contains the extended coil regions which are found even in the C-terminal domain where the luciferase active site is located [14,34]. Furthermore, the intrinsic disordered regions in a catalytically important part of luciferase may also be present.…”

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

“…In the absence of knowledge of the spatial structure of copepod luciferases, random and site‐directed mutagenesis is only the way to both improve catalytic properties of the enzyme and localize the amino acid residues involved in the formation of its active center. An attempt to determine the amino acids involved in the catalytic oxidation of the substrate and the formation of the active center using site‐directed mutagenesis was undertaken for MLuc7 luciferase . The internal cavity of hydromedusan Ca 2+ ‐regulated photoproteins which also use coelenterazine as a substrate of bioluminescence reaction contains many aromatic residues .…”