Safety evaluation of ß-agarase preparations from Streptomyces coelicolor A3(2).

Recent studies on neoagarooligosaccharides prepared by hydrolyzing agar with ß-agarase DagA produced from Streptomyces coelicolor A3(2) have enhanced our knowledge about the enzymatic utility of S. coelicolor. For safety evaluation, a crude extracellular protein containing DagA (crDagA) was prepared from the culture broth of S. coelicolor A3(2) M22-2C43, a highly productive strain of DagA. All genotoxicity tests, such as bacterial reverse mutation assay, eukaryotic chromosomal aberration assay, and in vivo micronucleus assay in mice showed no mutagenic activity of crDagA. No abnormalities were found in the appearance or behavior upon single oral administration up to 20,000 mg/kg body weight (BW) [318 mg TOS (Total Organic Solids)/kg BW] and long-term repeated oral administration toxicity tests up to 10,000 mg/kg BW/day (159 mg TOS/kg BW/day) in Sprague Dawley®™ rats. In addition, there were no statistically significant differences in the body weight change, food intake, hematology, blood biochemistry, organ weight, and clinical signs between the crDagA-administered and non-administered groups during the experimental period. This result showed that crDagA produced from S. coelicolor A3(2) is a safe, non-toxic substance, and therefore, can be used safely for manufacturing neoagarooligosaccharide, a functional substance effective in improving metabolic syndrome.

An Innovative Inhibitor with a New Chemical Moiety Aimed at Biliverdin IXß Reductase for Thrombocytopenia and Resilient against Cellular Degradation.

Biliverdin IXß reductase (BLVRB) has emerged as a promising therapeutic target for thrombocytopenia due to its involvement in reactive oxygen species (ROS) mechanisms. During the pursuit of inhibitors targeting BLVRB, olsalazine (OSA) became apparent as one of the most potent candidates. However, the direct application of OSA as a BLVRB inhibitor faces challenges, as it is prone to degradation into 5-aminosalicylic acid through cleavage of the diazenyl bond by abundant azoreductase (AzoR) enzymes in gut microbiota and eukaryotic cells. To overcome this obstacle, we devised olsalkene (OSK), an inhibitor where the diazenyl bond in OSA has been substituted with an alkene bond. OSK not only matches the efficacy of OSA but also demonstrates improved stability against degradation by AzoR, presenting a promising solution to this limitation. Furthermore, we have found that both OSK and OSA inhibit BLVRB, regardless of the presence of nicotinamide adenine dinucleotide phosphate, unlike other known inhibitors. This discovery opens new avenues for investigating the roles of BLVRB in blood disorders, including thrombocytopenia.

Kinetic evidence in favor of glyoxalase III and against deglycase activity of DJ-1.

DJ-1, a protein encoded by PARK7 plays a protective role against neurodegeneration. Since its glyoxalase III activity catalyzing methylglyoxal (MG) to lactate was discovered, DJ-1 has been re-established as a deglycase decomposing the MG-intermediates with amino acids and nucleotides (hemithioacetal and hemiaminal) rather than MG itself, but it is still debatable. Here, we have clarified that human DJ-1 directly recognizes MG, and not MG-intermediates, by monitoring the detailed catalytic processes and enantiomeric lactate products. The hemithioacetal intermediate between C106 of 15 N-labeled DJ-1 (15N DJ-1) and MG was also monitored by NMR. TRIS molecule formed stable diastereotopic complexes with MG (Kd , 1.57 ± 0.27 mM) by utilizing its three OH groups, which likely disturbed the assay of deglycase activity. The low kcat of DJ-1 for MG and its MG-induced structural perturbation may suggest that DJ-1 has a regulatory function as an in vivo sensor of reactive carbonyl stress.