Masaki Kuse

Synthesis of 13C-Dehydrocoelenterazine and NMR Studies on the Bioluminescence of a Symplectoteuthis Model

Abstract The bioluminescence of luminous squid (Symplectoteuthis oualaniensis) is presumed to be initiated by the addition of the sulfhydryl residue of a photoprotein to dehydrocoelenterazine (DCL). To clarify this step, a novel synthetic route was established to label DCL with 13C. Dithiothreitol (DTT) and glutathione (GSH) were used as photoprotein models. The addition of DTT and GSH to 13C-labeled DCL gave luminous chromophores. Its structures were confirmed by NMR and MS spectrometry. The DTT adduct emitted light under alkaline condition to produce an oxidized compound. Thus we succeeded in modeling the bioluminescence of a photoprotein with DTT.

Synthesis of 13C-dehydrocoelenterazine and model studies on Symplectoteuthis squid bioluminescence

In the photoprotein of an Okinawan squid bioluminescence of Symplectoteuthis oualaniensis L a dehydrocoelenterazine has been assigned as a chromophoric precursor to its apoprotein. To prove this mechanism, we have established new synthetic route to ca. 100%-13C incorporated dehydrocoelenterazine and coelenterazine at the neighboring carbon of the 2-position of 2,3-dihydroimidazo-[1,2a]-pyrazinone skeleton. This 13C enriched dehydrocoelenterazine readily converted in equilibrium between its adduct forms as a diastereomixture with glutathione (GSH) or dithiothreitol (DTT) compounds having sulfhydryl group. Structures of such adducts were fixed under acidic conditions and then discussed by NMR spectroscopy as well as absorbance and fluorescence spectra.

A novel photoprotein from oceanic squid (Symplectoteuthis oualaniensis) with sequence similarity to mammalian carbon-nitrogen hydrolase domains.

A 60-kDa photoprotein was selectively extracted from squid photogenic organ with 0.6 M KCl solution at pH 6 as luminescence-active forms. The photoprotein with fluorescence chromophore was eluted from size-exclusion HPLC mainly as oligomeric forms (about 200 kDa or more) with a trace amount of monomeric form of about 60 kDa. A limited tryptic digestion of the KCl-extract induced the cleavage into a 40-kDa fragment and a 16-kDa N-terminal fragment and the conversion to the monomeric form which still retained luminescence activity. Under UV light the 60-kDa protein and its 40-kDa fragment emitted fluorescence. Immunoblot analysis using specific antibody showed specific expression of the 60-kDa protein in the photogenic organ. Amino acid sequences of the 60-kDa photoprotein, its 40- and 16-kDa fragments, and six peptides from the Lys-C digest revealed no sequence similarity to known photoproteins but significant similarity to the carbon-nitrogen hydrolase domain found in mammalian biotinidase and vanin (pantetheinase).

Complexation of cyclic dodecadepsipeptide, cereulide with ammonium salts.

Cereulide is a principal toxin causing emetic syndrome which is produced by Bacillus cereus and has been known as potassium selective ionophore. This paper deals with its complexation with inorganic and organic ammonium ions to assign the higher structures similar to the complex with potassium ion by means of NMR and ESI-MS spectroscopy. Of particular interest, the detectable ions are not only at m/z 1191.8 as K(+) complex but also (or sometimes exclusively) at m/z 1170.8 as NH(4)(+) complex in its LC-MS analyses depending upon the conditions. This difference is due to the sample preparation and measurement condition.

7,8-Dihydropterin-6-carboxylic Acid as Light Emitter of Luminous Millipede, Luminodesmus sequoiae

A luminous millipede. Luminodesmus sequoiae, emits light centered at a wavelength of 500 nm. To determine the light emitter of this bioluminescent system, fluorescent compounds were isolated from pulverized cuticles. NMR and MS spectra of these compounds showed them to be pterin derivatives. Furthermore, proton/deuterium (H/D) exchange experiments by ESI-Q-TOF-MS and -MS/MS measurements have proved to be a powerful tool for elucidating these heteroaromatic compounds. Finally, we have concluded that 7,8-dihydropterin-6-carboxylic acid, a new natural product, is the light emitter of Luminodesmus bioluminescence.

High incorporation of l-amino acids to cereulide, an emetic toxin from Bacillus cereus

Cereulide is a principal toxin causing emetic syndrome produced by Bacillus cereus. This paper deals with biosynthetic studies on this unusual cyclic depsipeptide toxin from 13C labeled L-amino acid precursors (Val, Leu, Ala) upon cultivation in synthetic media. The analyses were made at atomic level of the constituent amino- or oxy-acids through NMR and ESI-MS/MS spectroscopic methods on cereulide and its hydrolysate dipeptides. The incorporation of the 13C atom was 95% in each O-Val, O-Leu and L-Val, while 40% in D-Ala of cereulide.

19F-Dehydrocoelenterazine as probe to investigate the active site of symplectin

Abstract Fluorinated dehydrocoelenterazines (F-DCTs) were synthesized to study molecular mechanisms of symplectin; a photoprotein of luminous squid Symplectoteuthis oualaniensis L. F-DCTs reacted with dithiothreitol and glutathione under neutral conditions to give the stable chromophores as symplectin model. Reconstructed symplectin was also obtained by addition of F-DCTs into apo-symplectin, and showed bioluminescence to emit 50–65% amount of light as natural symplectin. The structure of the chromophores was determined by 19F NMR, Q-TOF-MS, and MS/MS analyses. Sequencing of the chromopeptides of symplectin models prepared from F-DCTs and thiol compounds was accomplished by ESI-Q-TOF-MS/MS analysis.

Prevention of Copper-Induced Calcium Influx and Cell Death by Prion-Derived Peptide in Suspension-Cultured Tobacco Cells

Impact of copper on the oxidative and calcium signal transductions leading to cell death in plant cells and the effects of the copper-binding peptide derived from the human prion protein (PrP) as a novel plant-protecting agent were assessed using a cell suspension culture of transgenic tobacco (Nicotiana tabacum L., cell line BY-2) expressing the aequorin gene. Copper induces a series of biological and chemical reactions in plant cells including the oxidative burst reflecting the production of reactive oxygen species (ROS), such as hydroxyl radicals, and stimulation of calcium channel opening, allowing a transient increase in cytosolic calcium concentrations. The former was proven by the action of specifiC ROS scavengers blocking the calcium responses and the latter was proven by an increase in aequorin luminescence and its inhibition by specifi c channel blockers. Following these early events completed within 10 min, the development of copper-induced cell death was observed during additional 1 h in a dose-dependent manner. Addition of a synthetic peptide (KTNMKHMA) corresponding to the neurotoxic sequence in human PrP, prior to the addition of copper, effectively blocked both calcium influx and cell death induced by copper. Lastly, a possible mechanism of peptide action and future applications of this peptide in the protection of plant roots from metal toxicity or in favour of phytoremediation processes are discussed

Dehydrocoelenterazine is the Organic Substance Constituting the Prosthetic Group of Pholasin

The bivalve mollusc Pholas dactylus uses the luciferin–luciferase reaction for light emission. Its luciferin molecule is known as Pholasin, 2] a glycoprotein with a prosthetic group that is responsible for light emission. The luciferase molecule is a copper-containing peroxidase that catalyzes the oxidation of Pholasin to emit light in the presence of peroxides. The amount of light emission is proportional to the amount of the photoprotein Pholasin present. Henry and Michelson found that the superoxide radical produced by the reaction of luciferase and peroxides catalyzes the oxidation of luciferin. Because of their finding, it is now possible to initiate the light emission of Pholasin in a test tube in the presence of reactive oxygen species (ROS) without luciferase. Pholasin is now commercially available as an indicator of ROS. Despite the long research history of Pholasin (Dubois discovered a luciferin–luciferase reaction in the mollusc 122 years ago), the structure of its prosthetic group is unclear. Although Pholasin has been cloned, the expressed molecule lacks the prosthetic group and thus it cannot be activated in the same way as natural Pholasin. Elucidation of the prosthetic group structure would enable Pholasin to be widely used in many disciplines following simple activation by the addition of the organic substance. ROS are involved in many biological events, and thus selective monitoring of ROS is an important tool that would be more easily attainable by the commercial availability of Pholasin. We recently noticed a similarity between Pholasin and the oceanic squid Symplectoteuthis photoproteins: both emit strong light following the addition of a mixture of hydrogen peroxide and catalase or peroxidase. Symplectin, a photoprotein of S. oualaniensis, contains 501 amino acids, with eleven cysteines and a prosthetic group composed of dehydrocoelenterazine (DCL) bound to the eleventh cysteine (Cys390). Pholasin has half the number of amino acids as symplectin, and has five cysteines; the existence of one cysteine in free thiol form could constitute a prosthetic group by binding with DCL. We therefore hypothesized that the prosthetic group of Pholasin might be constituted from apoPholasin (Pholasin lacking a prosthetic group) and DCL (Scheme 1). Indeed, we previously reported that DCL could be used as an organic substance for Pholasin luminescence, although it is conceivable that it was mimicking the real organic substance. In the present study, we attempted to isolate DCL from Pholasin and show that it is the organic substance that forms the prosthetic group of Pholasin. DCL alone is not able to emit light even in the presence of ROS, however, after reacting with a thiol compound, the resulting DCL–thiol adduct then reacts with oxygen to emit light. The addition of DCL to a thiol compound forms an equilibrium, and the adduct might be stabilized in a photoprotein (Scheme 1). It was expected that extraction of DCL directly from Pholasin would be problematic as no liberated DCL

Identification of (Z)-3:(E)-2-Hexenal Isomerases Essential to the Production of the Leaf Aldehyde in Plants

The green odor of plants is characterized by green leaf volatiles (GLVs) composed of C6 compounds. GLVs are biosynthesized from polyunsaturated fatty acids in thylakoid membranes by a series of enzymes. A representative member of GLVs (E)-2-hexenal, known as the leaf aldehyde, has been assumed to be produced by isomerization from (Z)-3-hexenal in the biosynthesis pathway; however, the enzyme has not yet been identified. In this study, we purified the (Z)-3:(E)-2-hexenal isomerase (HI) from paprika fruits and showed that various plant species have homologous HIs. Purified HI is a homotrimeric protein of 110 kDa composed of 35-kDa subunits and shows high activity at acidic and neutral pH values. Phylogenetic analysis showed that HIs belong to the cupin superfamily, and at least three catalytic amino acids (His, Lys, Tyr) are conserved in HIs of various plant species. Enzymatic isomerization of (Z)-3-hexenal in the presence of deuterium oxide resulted in the introduction of deuterium at the C4 position of (E)-2-hexenal, and a suicide substrate 3-hexyn-1-al inhibited HI irreversibly, suggesting that the catalytic mode of HI is a keto-enol tautomerism reaction mode mediated by a catalytic His residue. The gene expression of HIs in Solanaceae plants was enhanced in specific developmental stages and by wounding treatment. Transgenic tomato plants overexpressing paprika HI accumulated (E)-2-hexenal in contrast to wild-type tomato plants mainly accumulating (Z)-3-hexenal, suggesting that HI plays a key role in the production of (E)-2-hexenal in planta.