Satoshi Inouye

The crystal structure of the photoprotein aequorin at 2.3 A resolution.

Aequorin is a calcium-sensitive photoprotein originally obtained from the jellyfish Aequorea aequorea. Because it has a high sensitivity to calcium ions and is biologically harmless, aequorin is widely used as a probe to monitor intracellular levels of free calcium. The aequorin molecule contains four helix–loop–helix ‘EF-hand’ domains, of which three can bind calcium. The molecule also contains coelenterazine as its chromophoric ligand. When calcium is added, the protein complex decomposes into apoaequorin, coelenteramide and CO2, accompanied by the emission of light. Apoaequorin can be regenerated into active aequorin in the absence of calcium by incubation with coelenterazine, oxygen and a thiol agent. Cloning and expression of the complementary DNA for aequorin were first reported in 1985 (refs 2, 6), and growth of crystals of the recombinant protein has been described; however, techniques have only recently been developed to prepare recombinant aequorin of the highest purity, permitting a full crystallographic study. Here we report the structure of recombinant aequorin determined by X-ray crystallography. Aequorin is found to be a globular molecule containing a hydrophobic core cavity that accommodates the ligand coelenterazine-2-hydroperoxide. The structure shows protein components stabilizing the peroxide and suggests a mechanism by which calcium activation may occur.

Site-specific cleavage of double-strand DNA by hydroperoxide of linoleic acid

The breakage of double‐strand (ds) DNA by 13‐L‐hydroperoxy‐cis‐9, trans‐11‐octadecadienoic acid (LAHPO) was investigated by agarose gel electrophoresis of supercoiled pBR322 DNA and the site of cleavage on the DNA molecule was determined by the method of DNA sequence analysis using 3‐end and 5‐end‐labeled DNA fragments as substrates. LAHPO caused cleavage at the position of guanine nucleotide in dsDNA. LAHPO caused dsDNA breaks at specific sites, but linoleic acid (LA) and 13‐L‐hydoxy‐cis‐9,trans‐11‐octadecadienoic acid (LAHO) have no such effects on dsDNA. The active oxygen atom of the hydroperoxy group of LAHPO was perhaps responsible for the site‐specific cleavage of dsDNA.

Firefly luciferase is a bifunctional enzyme: ATP‐dependent monooxygenase and a long chain fatty acyl‐CoA synthetase

Firefly luciferase can catalyze the formation of fatty acyl‐CoA via fatty acyl‐adenylate from fatty acid in the presence of ATP, Mg2+ and coenzyme A (CoA). A long chain fatty acyl‐CoA (C16–C20), produced by luciferase from a North American firefly (Photinus pyralis) and a Japanese firefly (Luciola cruciata), was isolated and identified by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry analysis. Of a number of substrates tested, linolenic acid (C18:3) and arachidonic acid (C20:4) appear to be suitable for acyl‐CoA synthesis. This evidence suggests that firefly luciferase within peroxisomes of the cells in the photogenic organ may be a bifunctional enzyme, catalyzing not only the bioluminescence reaction but also the fatty acyl‐CoA synthetic reaction.

The nucleotide sequences of copia and copia-related RNA in Drosophila virus-like particles

We have shown previously that Drosophila cells contain virus-like particles (VLPs) containing 5-kilobase (kb) RNA that hybridizes to a transposable element, termed copia. We have suggested that VLPs and copia are derivatives of viral particles and proviral forms, respectively, of ‘copia’ retrovirus, a putative Drosophila retrovirus1. To further clarify the relationship between copia and copia-related RNA in VLPs (VLP H-RNA), we determined and compared their nucleotide sequences. VLP H-RNA was found to be an unspliced, genome-sized transcript of copia, and, like retroviral genome RNA, VLP H-RNA is terminally redundant with termini localized in the long terminal repeats (LTRs) of copia. VLP H-RNA contains two long open reading frames (ORFs), one of which includes the coding sequence for a predominant VLP protein of relative molecular mass (Mr) 31,000 (31K). Here we show that, in contrast to 17.6 ORF2 (ref. 2), ORFs of copia have no extensive amino-acid sequence homology to the RT region2 of the reverse transcriptase of retrovirus in vertebrates. Because of a one-base insertion/deletion, the two ORFs in VLP H-RNA are fused and become a single, longer ORF in a genomic copia.

The crystal structures of semi-synthetic aequorins

The photoprotein aequorin emits light by an intramolecular reaction in the presence of a trace amount of Ca2+. Semi‐synthetic aequorins, produced by replacing the coelenterazine moiety in aequorin with the analogues of coelenterazine, show widely different sensitivities to Ca2+. To understand the structural basis of the Ca2+‐sensitivity, we determined the crystal structures of four semi‐synthetic aequorins (cp‐, i‐, br‐ and n‐aequorins) at resolutions of 1.6–1.8 Å. In general, the protein structures of these semi‐synthetic aequorins are almost identical to native aequorin. Of the four EF‐hand domains in the molecule, EF‐hand II does not bind Ca2+, and the loop of EF‐hand IV is clearly deformed. It is most likely that the binding of Ca2+ with EF‐hands I and III triggers luminescence. Although little difference was found in the overall structures of aequorins investigated, some significant differences were found in the interactions between the substituents of coelenterazine moiety and the amino acid residues in the binding pocket. The coelenterazine moieties in i‐, br‐, and n‐aequorins have bulky 2‐substitutions, which can interfere with the conformational changes of protein structure that follow the binding of Ca2+ to aequorin. In cp‐aequorin, the cyclopentylmethyl group that substitutes for the original 8‐benzyl group does not interact hydrophobically with the protein part, giving the coelenterazine moiety more conformational freedom to promote the light‐emitting reaction. The differences of various semi‐synthetic aequorins in Ca2+‐sensitivity and reaction rate are explained by the capability of the involved groups and structures to undergo conformational changes in response to the Ca2+‐binding.

Identification of biotinylated lysine residues in the photoprotein aequorin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mapping after lysine-specific endopeptidase digestion.

A method for identifying modified lysine residues in a protein, using lysine-specific endopeptidase treatment followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mapping, is described. As a model protein, the photoprotein aequorin was chosen and the N-hydroxysuccinimide ester of biotin was employed to chemically modify the lysine residues. After digestion with lysine-specific endopeptidase, the biotinylated residues of an amino terminus and five potential lysine residues were identified by MALDI-TOF-MS without any other separation procedure.

Isolation and properties of the luciferase stored in the ovary of the scyphozoan medusa Periphylla periphylla.

Bioluminescence of the medusa Periphylla is based on the oxidation of coelenterazine catalyzed by luciferase. Periphylla has two types of luciferase: the soluble form luciferase L, which causes the exumbrellar bioluminescence display of the medusa, and the insoluble aggregated form, which is stored as particulate material in the ovary, in an amount over 100 times that of luciferase L. The eggs are especially rich in the insoluble luciferase, which drastically decreases upon fertilization. The insoluble form could be solubilized by 2-mercaptoethanol, yielding a mixture of luciferase oligomers with molecular masses in multiples of approximately 20 kDa. Those having the molecular masses of 20 kDa, 40 kDa, and 80 kDa were isolated and designated, respectively, as luciferase A, luciferase B, and luciferase C. The luminescence activities of Periphylla luciferases A, B, and C were 1.2∼4.1 × 1016 photon/mgu2009·u2009s, significantly higher than any coelenterazine luciferase known, and the quantum yields of coelenterazine catalyzed by these luciferases (about 0.30 at 24u2009°C) are comparable to that catalyzed by Oplophorus luciferase (0.34 at 22u2009°C), which has been considered the most efficient coelenterazine luciferase until now. Luciferase L (32 kDa) could also be split by 2-mercaptoethanol into luciferase A and an accessory protein (approx. 12 kDa), as yet uncharacterized. Luciferases A, B, and C are highly resistant to inactivation: their luminescence activities are only slightly diminished at pH 1 and pH 11 and are enhanced in the presence of 1∼2 M guanidine hydrochloride; but they are less stable to heating than luciferase L, which is practically unaffected by boiling.

Chemical studies on the chiral indanone derivatives as the inhibitor of Renilla luciferase

Abstract The bioluminescence reaction of coelenterazine involves an oxidative process. To investigate the reaction mechanism, we synthesized three mechanism-based inhibitors with an indanone core structure. The inhibitors exhibited the competitive inhibition of the Renilla luciferase reaction. The (−)-4-benzyl-2-(4-hydroxybenzyl)-2-hydroxymethyl-6-(4-hydroxyphenyl)-indan-1-one showed the significant enantio-selectivity of the inhibition and its absolute configuration was assigned as the R -configuration. These inhibitors could be useful probes to study the catalytic environment in the coelenterazine–luciferase reaction.