Jun Hiratake

Structural basis for the spectral difference in luciferase bioluminescence.

Fireflies communicate with each other by emitting yellow-green to yellow-orange brilliant light. The bioluminescence reaction, which uses luciferin, Mg-ATP and molecular oxygen to yield an electronically excited oxyluciferin species, is carried out by the enzyme luciferase. Visible light is emitted during relaxation of excited oxyluciferin to its ground state. The high quantum yield of the luciferin/luciferase reaction and the change in bioluminescence colour caused by subtle structural differences in luciferase have attracted much research interest. In fact, a single amino acid substitution in luciferase changes the emission colour from yellow-green to red. Although the crystal structure of luciferase from the North American firefly (Photinus pyralis) has been described, the detailed mechanism for the bioluminescence colour change is still unclear. Here we report the crystal structures of wild-type and red mutant (S286N) luciferases from the Japanese Genji-botaru (Luciola cruciata) in complex with a high-energy intermediate analogue, 5′-O-[N-(dehydroluciferyl)-sulfamoyl]adenosine (DLSA). Comparing these structures to those of the wild-type luciferase complexed with AMP plus oxyluciferin (products) reveals a significant conformational change in the wild-type enzyme but not in the red mutant. This conformational change involves movement of the hydrophobic side chain of Ile 288 towards the benzothiazole ring of DLSA. Our results indicate that the degree of molecular rigidity of the excited state of oxyluciferin, which is controlled by a transient movement of Ile 288, determines the colour of bioluminescence during the emission reaction.

Identification and Molecular Cloning of a Novel Glycoside Hydrolase Family of Core 1 Type O-Glycan-specific Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum

We found endo-α-N-acetylgalactosaminidase in most bifidobacterial strains, which are predominant bacteria in the human colon. This enzyme catalyzes the liberation of galactosyl β1,3-N-acetyl-d-galactosamine (Galβ1,3GalNAc) α-linked to serine or threonine residues from mucin-type glycoproteins. The gene (engBF) encoding the enzyme has been cloned from Bifidobacterium longum JCM 1217. The protein consisted of 1,966 amino acid residues, and the central domain (590–1381 amino acid residues) exhibited 31–53% identity to hypothetical proteins of several bacteria including Clostridium perfringens and Streptococcus pneumoniae. The recombinant protein expressed in Escherichia coli liberated Galβ1,3GalNAc disaccharide from Galβ1,3GalNAcα1pNP and asialofetuin, but did not release GalNAc, Galβ1,3(GlcNAcβ1,6)GalNAc, GlcNAcβ1,3GalNAc, and Galβ1,3GlcNAc from each p-nitrophenyl (pNP) substrate, and also did not release sialo-oligosaccharides from fetuin, indicating its strict substrate specificity for the Core 1-type structure. The stereochemical course of hydrolysis was determined by 1H NMR and was found to be retention. Site-directed mutagenesis of a total of 22 conserved Asp and Glu residues suggested that Asp-682 and Asp-789 are critical residues for the catalytic activity of the enzyme. The enzyme also exhibited transglycosylation activity toward various mono- and disaccharides and 1-alkanols, demonstrating its potential to synthesize neoglycoconjugates. This is the first report for the isolation of a gene encoding endo-α-N-acetylgalactosaminidase from any organisms and for the establishment of a new glycoside hydrolase family (GH family 101).

Structural basis for gating mechanisms of a eukaryotic P-glycoprotein homolog.

Significance P-glycoprotein exports various hydrophobic chemicals in an ATP-dependent manner, determines their absorption and distribution in the body, and is involved in multidrug resistance (MDR) in tumors. Understanding the mechanism of the multidrug transport is important for designing drugs of good bioavailability and efficient cancer chemotherapy. We determined the high-resolution crystal structures of a eukaryotic P-glycoprotein homolog and revealed the detailed architecture of its transmembrane domains, which contain an exit gate for substrates that opens to the extracellular side and two entrance gates that open to the intramembranous region and the cytosolic side. We propose a motion of the transmembrane domains powered by the association of two nucleotide-binding domains on ATP binding that is different from other transporters. P-glycoprotein is an ATP-binding cassette multidrug transporter that actively transports chemically diverse substrates across the lipid bilayer. The precise molecular mechanism underlying transport is not fully understood. Here, we present crystal structures of a eukaryotic P-glycoprotein homolog, CmABCB1 from Cyanidioschyzon merolae, in two forms: unbound at 2.6-Å resolution and bound to a unique allosteric inhibitor at 2.4-Å resolution. The inhibitor clamps the transmembrane helices from the outside, fixing the CmABCB1 structure in an inward-open conformation similar to the unbound structure, confirming that an outward-opening motion is required for ATP hydrolysis cycle. These structures, along with site-directed mutagenesis and transporter activity measurements, reveal the detailed architecture of the transporter, including a gate that opens to extracellular side and two gates that open to intramembranous region and the cytosolic side. We propose that the motion of the nucleotide-binding domain drives those gating apparatuses via two short intracellular helices, IH1 and IH2, and two transmembrane helices, TM2 and TM5.

Sensitive β-galactosidase-targeting fluorescence probe for visualizing small peritoneal metastatic tumours in vivo.

Fluorescence-guided diagnostics is one of the most promising approaches for facile detection of cancer in situ. Here we focus on β-galactosidase, which is overexpressed in primary ovarian cancers, as a molecular target for visualizing peritoneal metastases from ovarian cancers. As existing fluorescence probes are unsuitable, we have designed membrane-permeable HMRef-βGal, in which the optimized intramolecular spirocyclic function affords >1,400-fold fluorescence enhancement on activation. We confirm that HMRef-βGal sensitively detects intracellular β-galactosidase activity in several ovarian cancer lines. In vivo, this probe visualizes metastases as small as <1 mm in diameter in seven mouse models of disseminated human peritoneal ovarian cancer (SHIN3, SKOV3, OVK18, OVCAR3, OVCAR4, OVCAR5 and OVCAR8). Because of its high brightness, real-time detection of metastases with the naked eye is possible. Endoscopic fluorescence detection of metastases is also demonstrated. The results clearly indicate preclinical potential value of the probe for fluorescence-guided diagnosis of peritoneal metastases from ovarian cancers.

Substrate Specificity of β-Primeverosidase, A Key Enzyme in Aroma Formation during Oolong Tea and Black Tea Manufacturing

We synthesized nine kinds of diglycosides and a monoglycoside of 2-phenylethanol to investigate the substrate specificity of the purified β-primeverosidase from fresh leaves of a tea cultivar (Camellia sinensis var. sinensis cv. Yabukita) in comparison with the apparent substrate specificity of the crude enzyme extract from tea leaves. The crude enzyme extract mainly showed β-primeverosidase activity, although monoglycosidases activity was present to some extent. The purified β-primeverosidase showed very narrow substrate specificity with respect to the glycon moiety, and especially prominent specificity for the β-primeverosyl (6-O-β-D-xylopyranosyl-β-D-glucopyranosyl) moiety. The enzymes hydrolyzed naturally occurring diglycosides such as β-primeveroside, β-vicianoside, β-acuminoside, β-gentiobioside and 6-O-α-L-arabinofuranosyl-β-D-glucopyranoside, but were unable to hydrolyze synthetic unnatural diglycosides. The purified enzyme was inactive toward 2-phenylethyl β-D-glucopyranoside. The enzyme hydrolyzed each of the diglycosides into the corresponding disaccharide and 2-phenylethanol. These results indicate the β-primeverosidase, a diglycosidase, to be a key enzyme involved in aroma formation during the tea manufacturing process.

Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 β-N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core

Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a β-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a β-N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary N-glycan substrate, it exhibited a 5-fold preference for removal of the β(1,2)-linked N-acetylglucosamine from the Manα(1,3) branch compared with the Manα(1,6) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic analysis suggested that Sfhex is an ortholog of mammalian lysosomal β-N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells, a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this study suggests that Sfhex may play a role as a processing β-N-acetylhexosaminidase acting on N-glycans from Sf9 cells.

β-d-Glycosylamidines: potent, selective, and easily accessible β-glycosidase inhibitors

Abstract β- d -Glycosylamidines, in which a glycon is connected via an N -glycoside linkage with a substituted amidine (aglycon), were synthesized in two steps from the corresponding sugars and served as stable and potent β-glycosidase inhibitors with high selectivity according to the glycon- and α, β-specificities of the enzymes.

Enantiotopic-group differentiation. Catalytic asymmetric ring-opening of prochiral cyclic acid anhydrides with methanol, using cinchona alkaloids

Asymmetric ring-opening of prochiral acid anhydrides (1) with methanol has been achieved by a catalytic quantity of cinchona alkaloids (2). The product, the optically active half-ester (3), has been subjected to functional-group-selective reduction to give the optically active lactones (5). The reaction rate of the ring-opening and the extent of selectivity are dependent on the nature of the reaction medium, the polarity of solvent, and substrate concentration. By selecting the reaction conditions, an enantiometric excess of up to 70% has been obtained. The kinetic isotope effect and other mechanistic investigations suggest that the reaction proceeds via general-base catalysis by the quinuclidine moiety of the base (2), and that the relative configuration of the C-9 hydroxy group with respect to the C-8 quinuclidine amino function determines the selectivity of the reaction.

Crystal Structures of Escherichia coli γ-Glutamyltranspeptidase in Complex with Azaserine and Acivicin: Novel Mechanistic Implication for Inhibition by Glutamine Antagonists

gamma-Glutamyltranspeptidase (GGT) catalyzes the cleavage of such gamma-glutamyl compounds as glutathione, and the transfer of their gamma-glutamyl group to water or to other amino acids and peptides. GGT is involved in a number of biological phenomena such as drug resistance and metastasis of cancer cells by detoxification of xenobiotics. Azaserine and acivicin are classical and irreversible inhibitors of GGT, but their binding sites and the inhibition mechanisms remain to be defined. We have determined the crystal structures of GGT from Escherichia coli in complex with azaserine and acivicin at 1.65 A resolution. Both inhibitors are bound to GGT at its substrate-binding pocket in a manner similar to that observed previously with the gamma-glutamyl-enzyme intermediate. They form a covalent bond with the O(gamma) atom of Thr391, the catalytic residue of GGT. Their alpha-carboxy and alpha-amino groups are recognized by extensive hydrogen bonding and charge interactions with the residues that are conserved among GGT orthologs. The two amido nitrogen atoms of Gly483 and Gly484, which form the oxyanion hole, interact with the inhibitors directly or via a water molecule. Notably, in the azaserine complex the carbon atom that forms a covalent bond with Thr391 is sp(3)-hybridized, suggesting that the carbonyl of azaserine is attacked by Thr391 to form a tetrahedral intermediate, which is stabilized by the oxyanion hole. Furthermore, when acivicin is bound to GGT, a migration of the single and double bonds occurs in its dihydroisoxazole ring. The structural characteristics presented here imply that the unprecedented binding modes of azaserine and acivicin are conserved in all GGTs from bacteria to mammals and give a new insight into the inhibition mechanism of glutamine amidotransferases by these glutamine antagonists.

Preventive effect of GGsTop, a novel and selective γ-glutamyl transpeptidase inhibitor, on ischemia/reperfusion-induced renal injury in rats.

GGsTop [2-amino-4-{[3-(carboxymethyl)phenyl](methyl)phosphono}butanoic acid], is a novel, highly selective, and irreversible γ-glutamyl transpeptidase (GGT) inhibitor with no inhibitory activity on glutamine amidotransferases. In this study, we investigated the effects of treatment with GGsTop on ischemia/reperfusion-induced renal injury in uninephrectomized rats. Ischemic acute kidney injury (AKI) was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion 2 weeks after contralateral nephrectomy. Renal function in vehicle-treated AKI rats markedly decreased at 1 day after reperfusion. Treatment with GGsTop (1 and 10 mg/kg i.v.) 5 min before ischemia attenuated the ischemia/reperfusion-induced renal dysfunction in a dose-dependent manner. Histopathological examination of the kidney of AKI rats revealed severe renal damage, which was significantly suppressed by the GGsTop treatment. In renal tissues exposed to ischemia/reperfusion, GGT activity was markedly increased immediately after reperfusion, whereas renal superoxide production and malondialdehyde level were significantly increased 6 h after reperfusion. These alterations were abolished by the treatment with GGsTop. In addition, renal glutathione content was decreased by the 45-min ischemia, but its level was markedly elevated by the GGsTop treatment. Our results demonstrate that the novel and highly selective GGT inhibitor GGsTop prevents ischemia/reperfusion-induced AKI. The renoprotective effect of GGsTop seems to be attributed to the suppression of oxidative stress by inhibiting GGT activation, thereby preventing the degradation of glutathione.