Biology subjects

Kuramochi, K.

Publications and source records attributed to Kuramochi, K..

5 recordsLinked to original sources

bioRxiv2024

A beta-Galactosidase acting on unique galactosides: the structure and function of a beta-1,2-galactosidase from Bacteroides xylanisolvens, an intestinal bacterium

Galactosides are major carbohydrates that are found in plant cell walls and various prebiotic oligosaccharides. Studying the detailed biochemical functions of {beta}-galactosidases in degrading these carbohydrates is important. In particular, identifying {beta}-galactosidases with new substrate specificities could help in the production of potentially beneficial oligosaccharides. In this study, we identified a {beta}-galactosidase with novel substrate specificity from Bacteroides xylanisolvens, an intestinal bacterium. The enzyme did not show hydrolytic activity toward natural {beta}-galactosides during the first screening. However, when -D-galactosyl fluoride (-GalF) as a donor substrate and galactose or D-fucose as an acceptor substrate were incubated with a nucleophile mutant, reaction products were detected. The galactobiose produced from the -GalF and galactose was identified as {beta}-1,2-galactobiose using NMR. Kinetic analysis revealed that this enzyme effectively hydrolyzed {beta}-1,2-galactobiose and {beta}-1,2-galactotriose. In the complex structure with methyl {beta}-galactopyranose as a ligand, the ligand is only located at subsite +1. The 2-hydroxy group and the anomeric methyl group of methyl {beta}-galactopyranose faces in the direction of subsite -1 and the solvent, respectively. This observation is consistent with the substrate specificity of the enzyme regarding linkage position and chain length. Overall, we concluded that the enzyme is a {beta}-galactosidase acting on {beta}-1,2-galactooligosaccharides. SynopsisThe structural and functional analysis of {beta}-galactosidase from an intestinal bacterium led to the discovery of a new {beta}-galactosidase hydrolyzing unique {beta}-1,2-galactooligosaccharides.

biochemistry↗

bioRxiv2023

Functional and structural analysis of a cyclization domain in a cyclic beta-1,2-glucan synthase

Cyclic {beta}-1,2-glucan synthase (CGS) is a key enzyme in production of cyclic {beta}-1,2-glucans (C{beta}Gs) which are involved in bacterial infection or symbiosis to host organisms. Nevertheless, a mechanism of cyclization, the final step in the CGS reaction, has not been fully understood. Here we performed functional and structural analyses of the cyclization domain of CGS alone from Thermoanaerobacter italicus (TiCGSCy). We first found that {beta}-glucosidase-resistant compounds are produced by TiCGSCy with linear {beta}-1,2-glucans as substrates. The 1H-NMR analysis revealed that these products are C{beta}Gs. Next, action pattern analyses using {beta}-1,2-glucooligosaccharides revealed a unique reaction pattern: exclusive transglycosylation without hydrolysis and a hexasaccharide as the minimum length of the substrate. Nevertheless, longer substrate {beta}-1,2-glucooligosaccharides are preferred, being consistent with the fact that CGSs generally produce C{beta}Gs with degrees of polymerization of around 20. Finally, the overall structure of the cyclization domain of TiCGSCy was found to be similar to those of {beta}-1,2-glucanases in phylogenetically different groups. Meanwhile, the identified catalytic residues indicated clear difference in the reaction pathways between these enzymes. Therefore, we propose a novel reaction mechanism of TiCGSCy. Thus, we propose that the present group of CGSs should be defined as a new glycoside hydrolase family, GHxxx. Statements and DeclarationsCompeting Interests: The authors declare no competing interests. Key PointsIt was clearly evidenced that cyclization domain alone produces cyclic {beta}-1,2-glucans. The domain exclusively catalyzes transglycosylation without hydrolysis. The present catalytic domain should be defined as a new glycoside hydrolase family.

biochemistry↗

bioRxiv2021

Identification of anti-severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) oxysterol derivatives in vitro

Development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) are urgently needed to combat the coronavirus disease 2019 (COVID-19). Oxysterols, defined as oxidized derivatives of cholesterol, include endogenous (naturally occurring) cholesterol metabolites as well as semi-synthetic oxysterol derivatives. We have previously studied the use of semi-synthetic oxysterol derivatives as drug candidates for inhibition of cancer, fibrosis, and bone regeneration. In this study, we have screened a panel of naturally occurring and semi-synthetic oxysterol derivatives for anti-SARS-CoV-2 activity, using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy186 displayed antiviral activity comparable to natural oxysterols. In addition, related oxysterol analogues Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 M and 99% at 15 M, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fall into a therapeutically relevant range (19 M), based on the dose-dependent curve for antiviral activity in our cell culture infection assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 with disrupting the formation of double membrane vesicles (DMVs), intracellular membrane compartments associated with viral replication. Oxy210 also inhibited the replication of hepatitis C virus, another RNA virus whose replication is associated with DMVs, but not the replication of the DMV-independent hepatitis D virus. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk developing COVID-19.

microbiology↗

bioRxiv2020

Anti-severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) potency of Mefloquine as an entry inhibitor in vitro

Coronavirus disease 2019 (COVID-19) has caused serious public health, social, and economic damage worldwide and effective drugs that prevent or cure COVID-19 are urgently needed. Approved drugs including Hydroxychloroquine, Remdesivir or Interferon were reported to inhibit the infection or propagation of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), however, their clinical efficacies have not yet been well demonstrated. To identify drugs with higher antiviral potency, we screened approved anti-parasitic/anti-protozoal drugs and identified an anti-malarial drug, Mefloquine, which showed the highest anti-SARS-CoV-2 activity among the tested compounds. Mefloquine showed higher anti-SARS-CoV-2 activity than Hydroxychloroquine in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 1.28 M, IC90 = 2.31 M, and IC99 = 4.39 M in VeroE6/TMPRSS2 cells. Mefloquine inhibited viral entry after viral attachment to the target cell. Combined treatment with Mefloquine and Nelfinavir, a replication inhibitor, showed synergistic antiviral activity. Our mathematical modeling based on the drug concentration in the lung predicted that Mefloquine administration at a standard treatment dosage could decline viral dynamics in patients, reduce cumulative viral load to 7% and shorten the time until virus elimination by 6.1 days. These data cumulatively underscore Mefloquine as an anti-SARS-CoV-2 entry inhibitor.

microbiology↗

bioRxiv2020

Multidrug treatment with nelfinavir and cepharanthine against COVID-19

Antiviral treatments targeting the emerging coronavirus disease 2019 (COVID-19) are urgently required. We screened a panel of already-approved drugs in a cell culture model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and identified two new antiviral agents: the HIV protease inhibitor Nelfinavir and the anti-inflammatory drug Cepharanthine. In silico modeling shows Nelfinavir binds the SARS-CoV-2 main protease consistent with its inhibition of viral replication, whilst Cepharanthine inhibits viral attachment and entry into cells. Consistent with their different modes of action, in vitro assays highlight a synergistic effect of this combined treatment to limit SARS-CoV-2 proliferation. Mathematical modeling in vitro antiviral activity coupled with the known pharmacokinetics for these drugs predicts that Nelfinavir will facilitate viral clearance. Combining Nelfinavir/Cepharanthine enhanced their predicted efficacy to control viral proliferation, to ameliorate both the progression of disease and risk of transmission. In summary, this study identifies a new multidrug combination treatment for COVID-19.

microbiology↗