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Metallo-β-lactamases (MBLs) hydrolyze a broad range of β-lactams, including carbapenems. VIM-28, an MBL identified in Pseudomonas aeruginosa, is an H224L/S228R variant of VIM-1 and H224L variant of VIM-4. Compared with VIM-26 (R228S), VIM-28 displayed decreased Km (12.5 for VIM-28 vs 513 μM for VIM-26; 9.66 vs 150 μM) and increased kcat/Km(15.3 vs 1.81 μM-1s-1; 28.6 vs 5.89 μM-1s-1) for ampicillin and cephalothin, respectively. VIM-1, which has a His in position 224 and Ser in position 228, displayed intermediate kinetic values (Km 215 and 77.0 μM; kcat/Km 2.63 and 8.61 μM-1s-1) for ampicillin and cephalothin, respectively, indicating that the presence of a positively charged residue at either position 224 or 228 enhanced substrate interactions. The combined L224H/R228S substitutions in VIM-1 increased the catalytic efficiency of the enzyme for ceftazidime by more than one order of magnitude. These kinetic trends were consistent with the minimum inhibitory concentration (MIC) data, with an eightfold increase in ceftazidime MIC for VIM-1-producing cells. Moreover, relative MIC assay showed that VIM-26 (R228S)-producing cells were more refractory to the addition of chelators than cells producing VIM-28, whereas VIM-4 (L224H)-producing cells showed reduced resistance, suggesting that the residues at positions 224 and 228 influence the metal-binding affinity of the enzyme. Differential scanning fluorimetry assay revealed that the R228S substitution increased the melting temperature of the enzyme, whereas the L224H substitution reduced its thermal stability. VIM-28 exhibited high catalytic efficiency for substrates other than ceftazidime, and the H224L substitution conferred higher zinc-binding affinity and thermal stability compared with VIM-4.IMPORTANCEβ-Lactam-resistant bacteria, especially carbapenem-resistant strains, pose a major global health threat, often through metallo-β-lactamases (MBLs). To anticipate resistance evolution, we characterized VIM-28, a variant of the widespread VIM-1/VIM-4-type enzymes, focusing on the roles of two variable L10 loop residues. Substitutions at positions 224 and 228 strongly affected substrate specificity, enzyme stability, and zinc affinity. Arg228 was important for carbapenem recognition, while combined substitutions at positions 224 and 228 could enhance activity toward ceftazidime. Notably, the R228S substitution improved zinc binding and thermal stability, supporting enzyme function under zinc-limited host conditions. These findings reveal mechanisms driving MBL diversity and highlight evolutionary strategies sustaining antibiotic resistance.
Abstract A complex of the 3′-truncated tRNA Arg that lacks 9 nt and tRNase Z L works as a GCCC-recognizing RNA cutter. It recognizes an RNA substrate via four Watson-Crick-Franklin base-pairings with the 3′-truncated tRNA Arg . Human SLFN11 and SLFN13 can generate 3′-truncated tRNA Leu that lacks 10 nt and 3′-truncated tRNA Ser that lacks 11 nt, respectively, from their corresponding mature tRNAs. Here, we investigated if these 3′-truncated tRNAs together with tRNase Z L work as sequence-specific RNA cutters. We examined five RNA targets for cleavage by recombinant human tRNase Z L in the presence of the 3′-truncated tRNA Leu or tRNA Ser . We demonstrated that the 3′-truncated tRNA Leu and tRNA Ser together with tRNase Z L indeed work as ∼6-base-recognizing and 7-base-recognizing RNA cutters, respectively.
Type 2 diabetes and osteoporosis are very serious diseases all over the world. We prepared noodles from ‘Kitanokaori’ (newly developed wheat) (KITs) using weakly acidic hard water, which showed a higher amount of resistant starch (9.0-fold) and calcium (2.7-fold) than noodles from Sanukinoyume (premium wheat) (SANs) using purified water. Furthermore, aged mice, which were fed a diet of KIT using weakly acidic hard water for eight weeks, showed lower postprandial blood glucose levels (BGLs) at 30 min after consumption than mice fed a control diet (SAN using purified water) (p < 0.05). Therefore, KIT seems promising in terms of health promotion through food. Additionally, the whiteness (WB) and brightness (L*) of wheat noodles using weakly acidic hard water showed higher values than ones using purified water. The texture of KIT using weakly acidic hard water showed few textural differences from noodles using purified water. The KIT using weakly acidic hard water would be acceptable in terms of palatability and bio-functionality in terms of delaying digestion.
The structural and bonding properties of liquid L -lactic acid were investigated through ab initio molecular dynamics simulations, leveraging on the agreement between the static structure factors obtained from high-energy X-ray di ff raction (HEXRD) measurements. It has been demonstrated that predominant intermolecular interactions arise from hydrogen bonding. Hydrogen atoms in two hydroxy groups tend to form hydrogen bonds with two of the three oxygen atoms in neighboring molecules. This results in the presence of an oxygen atom in the hydroxy group of the carboxy group that does not signi fi cantly contribute to hydrogen bond formation with neighboring hydrogen atoms. This is consistent with the behavior observed in the crystalline phase. However, in the liquid state, four types of intermolecular hydrogen bonds are primarily formed, whereas in the crystal, only two types are observed. We show that the directionality of hydrogen bonds determines the intermolecular orientation. We thoroughly studied the structural properties and covalent bonding between atoms and charged states of atoms.
Recently, global warming has led to an increase in chalky rice grains. This has consequently resulted in the deterioration in quality of rice products. Although we previously reported that hard water, rich in Ca, is useful for the quality improvement of high-temperature-damaged rice grains, the mechanism was not elucidated sufficiently. Therefore, we used various kinds of rice cultivars, from waxy to high-amylose ones, for soaking and boiling in hard water and compared physical and chemical properties of the products. It was shown that the degree of quality improvement, such as final viscosities in pasting property, and textural properties of boiled rice, was more remarkable for high-amylose rice than low-amylose rice. As we found that the phosphorus contents showed positive correlations with amylose and long chains of amylopectin, we estimate that the effects are mainly due to binding of calcium and phosphorus. Because that high-amylose or long-chain-rich amylopectin rice cultivars showed high calcium contents in rice products, these rice cultivars would be very useful to supply calcium through dietary intake via hard water cooking.
