Symplectin

Fig. 6.3.7 Luminescence spectrum of a homogenate of the luminous organ of Symplectoteuthis oualaniensis in the presence of 0.5 M KC1 (from Tsuji and Leisman, 1981). A homogenate suspension (1 ml) and 1MKC1 (1 ml), both made with 50 mM Tris-HCl, pH 7.6, containing 1 mM dithioerythritol, were mixed and the spectrum was measured 6 min after mixing. Note that the luminescence of the photoprotein symplectin isolated from the luminous organs showed a maximum at 470—480 nm (Takahashi and Isobe, 1993, 1994).

Fig. 6.3.8 Chemical structures of the compounds relevant to the luminescence reaction of symplectin. From Takahashi and Isobe, 1993.

Fig. 6.3.9 An illustration showing the mechanism of the reconstitution and luminescence of symplectin. The binding of dehydrocoelenterazine with the SH group of a cysteine residue of aposymplectin (left) results in the formation of active symplectin (center). Symplectin is oxygenated at the C2 position, resulting in the formation of coelenteramide bound to aposymplectin (right), accompanied by the emission of light. From Isobe et al., 2002, with permission from Elsevier.

The luminescence reaction of symplectin induced by warming is slow and not sufficiently bright to account for the bright luminescence of the live squid. The factors and conditions that cause the intense in vivo luminescence remain to be identified. 

Symplectoteuthis oualaniensis11 Symplectoteuthis luminosap Symplectin 60,000 50,000 Alkaline pH and O2 Catalase, H2O2 and 02 470-480°... 

Isobe, M., et al. (2002). 19F-Dehydrocoelenterazine as probe to investigate the active site of symplectin. Tetrahedron 58 2117-2126. 

Symplectin, 208-210, 347 isolation and purification, 208 molecular size and properties, 209 regeneration with... 

R653 M. Isobe, M. Kuse, T. Fujii, H. Takahashi, K. Ohshima, H. Mori, J.-Y. Ahn and M. Tsukasa, Molecular Mechanisms of Bioluminescence of Symplectin, a Photoprotein from Okinawan Squid, Symplectoteuthis Oualaniensis L , Tennen Yuki Kagobutsu Toronkai Koen Yoshishu, 2000, 42, 97... 

Now we focus on the structural analysis of symplectin active site and the molecular mechanism of symplectin bioluminescence. ... 

From the sequence analysis of symplectin with LC-Q-TOF-MS, MS/MS and cDNA, symplectin has 501 amino acids sequence. Partial degradation of symplectin with trypsin afforded a 40 kDa protein (symplectin A ), which is the C-terminal part of symplectin and still has bioluminescent activity. We suppose that the active site of symplectin exists in the 40 kDa symplectin A. ... 

C-labeled DCT analog C C-DCTa) was synthesized to prove the chromophoric structure of symplectin. Dithiothreitol and glutathione (apo-symplectin models) reacted with C-DCTa to afford the luminescent active chromophores, which were analyzed with NMR and MS. We demonstrated that DCT binds with the sulfhydryl residues at the 2 - C-labeled carbon as shown in Fig. 2, and we succeeded in reproducing a model symplectin bioluminescence with C-DCTa. ... 

We also found the equilibrium between DCT and thiol adducts that is the origin of the weakness of chromophoric C-S bond. To detect the symplectin active center cysteine, it was necessary to make the C-S bond tight. Therefore, we decided to introduce a fluorine atom into DCT 2 -aromatic ring instead of hydroxyl and methoxy group. 

Fluorinated dehydrocoelenterazine (F-DCT) was synthesized as a probe to investigate the active center cysteine of symplectin. We found that F-DCT strongly bound to the sulfhydryl residue of cysteine to afford a stable chromophore, which was proved with NMR and MS. There was also no equilibrium between F-DCT and its thol adducts as expected. Three F-DCTs (ortho, meta, para) were synthesized and... 

Reconstituted symplectin (Recon-symplectin) was prepared from 2-ortho- -DCT and apo-symplectin. The Recon-symplectin was proteolytically digested with trypsine to obtain a chromophoric peptide, which contains both the active center cysteine and F-DCT. Nano-LC-MS analysis afforded plausible data for the chromophoric peptide of the symplectin active center. But, we could not perfectly demonstrate the active center cysteine of symplectin with MS/MS analysis. 

Now we investigate the symplectin active site by using a photoaffinity labeling and the nano-LC-Q-TOF-MS analysis and also study the structure and activity relationship (SAR) between DCT structures and symplectin bioluminescent activities. For the SAR study, we also developed a novel synthetic method for DCT analogs. ... 

Trypsin digestion of reconstituted symplectin with F-DCL afforded peptides that bound to the active site cysteine (MS identification). However, these results were preliminary, because we could not obtain MS/MS data of these peptides.10,11 We therefore decided to synthesize two di-fluoro-DCLs (diF-DCL-1 and diF-DCL-2) that would make the chromophore structure more stable than F-DCLs and would afford increase of+18 Da compare to the corresponding F-DCLs peak.12... 

Working hypothesis. We now postulate that the absolute configuration of the chromophore in the symplectin active site might be important based on these results. Although both substrate (DCL) and product (amide compound and coelenteramine) have no chirality (Fig. 1), we now assume that dynamic chirality of the active site could play an important role in bioluminescence. We are now trying to analyze the reaction products in order to answer the question why diF-DCLs have different activity. 

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