Cold-active enzymes increase their catalytic efficiency at low-temperature, introducing structural flexibility at or near the active sites. Inevitably, this feat seems to be accompanied by lower thermal stability. These characteristics have made cold-active enzymes into attractive targets for the industrial applications, since they could reduce the energy cost in the reaction, attenuate side-reactions, and simply be inactivated. In addition, the increased structural flexibility could result in broad substrate specificity for various non-native substrates, which is called substrate promiscuity. In this perspective, we deal with a less addressed aspect of cold-active enzymes, substrate promiscuity, which has enormous potential for semi-synthesis or enzymatic modification of fine chemicals and drugs. Further structural and directed-evolutional studies on substrate promiscuity of cold-active enzymes will provide a new workhorse in white biotechnology.
Background: Bats have been considered natural reservoirs for several pathogenic human coronaviruses (CoVs) in the last two decades. Recently, a bat CoV was detected in the Republic of Korea; its entire genome was sequenced and reported to be genetically similar to that of the severe acute respiratory syndrome CoV (SARS-CoV). Objectives: The objective of this study was to compare the genetic sequences of SARS-CoV, SARS-CoV-2, and the two Korean bat CoV strains 16BO133 and B15-21, to estimate the likelihood of an interaction between the Korean bat CoVs and the human angiotensinconverting enzyme 2 (ACE2) receptor. Methods: The phylogenetic analysis was conducted with the maximum-likelihood (ML) method using MEGA 7 software. The Korean bat CoVs receptor binding domain (RBD) of the spike protein was analyzed by comparative homology modeling using the SWISS-MODEL server. The binding energies of the complexes were calculated using PRODIGY and MM/GBGA. Results: Phylogenetic analyses of the entire RNA-dependent RNA polymerase, spike regions, and the complete genome revealed that the Korean CoVs, along with SARS-CoV and SARS-CoV-2, belong to the subgenus Sarbecovirus, within BetaCoVs. However, the two Korean CoVs were distinct from SARS-CoV-2. Specifically, the spike gene of the Korean CoVs, which is involved in host infection, differed from that of SARS-CoV-2, showing only 66.8%-67.0% nucleotide homology and presented deletions within the RBD, particularly within regions critical for cross-species transmission and that mediate interaction with ACE2. Binding free energy calculation revealed that the binding affinity of Korean bat CoV RBD to hACE2 was drastically lower than that of SARS-CoV and SARS-CoV-2. Conclusions: These results suggest that Korean bat CoVs are unlikely to bind to the human ACE2 receptor.
To survive at low temperatures, psychrophiles seem to produce cold-adapted enzymes with a high flexibility around active sites for high catalytic efficiency. To gain insights into the cold-adaptation of psychrophilic enzymes in atomic detail, we determined the crystal structure of 5-enolpyruvylshikimate-3-phosphate synthase (CpsEPSPS) from Colwellia psychrerythraea, a psychrophilic bacterium. EPSPS is the primary target for the broad-spectrum herbicide, glyphosate, and a promising target for the development of antimicrobial and antiparasitic agents since it is absent in animals. The crystal structure of unliganded, open CpsEPSPS was determined at 2.2 Å resolution in space group P2 with two protomers per asymmetric unit. Superposition of separate domain I and II of CpsEPSPS structure with those of Escherichia coli EPSPS (EcoEPSPS) structure showed relatively small differences of RMSD values of 0.423 Å and 0.693 Å for domains I and II, respectively, implying the residues in ligand binding and catalysis of cold-adapted CpsEPSPS showed no significant flexibility. This result is conflicting to other cases of cold-adapted proteins. We also observed that hydrogen-bond forming residues in the surface of EcoEPSPS was mutated to non- or lesser hydrogen-bond forming one in CpsEPSPS, which makes the protein surface softer and eventually makes the protein more active at low temperature. In addition, domain rotation angle between open and closed states of CpsEPSPS was smaller than those of any EPSPSs whose structures are known. The restriction of the domain closure, which reduces the entropy cost of ligand binding and catalysis, may be a novel molecular adaptations of cold-adapted enzymes.
Prebiotics and probiotics have gained much attention in the pursuit of a healthy life. Recently, postbiotics have been spotlighted as next-generation compounds that can improve health. Postbiotics are designated into non-viable, inactivated, and ghost probiotics, and linked to several health benefits for the gut, immune system, and various other aspects of health. This study investigated the anti-proliferation effects of postbiotics against HT-29 cells, a colon cancer cell line. The postbiotics were produced by the ultrasonication method from two Lactobacillus strains (Lactobacillus sp. La1, and La2) and designated to Pobt-La1 and Pobt-La2, respectively, and non-viability was confirmed on the plate media. The anti-proliferation effect was concentration-dependent. The HT-29 cells showed viabilities of 39% and 49% when treated with 300 µL/mL of Pobt-La1 and Pobt-La2, respectively. During observation of the morphological changes of HT-29 cells when treated with IC50, a cell nucleus was not observed but cell condensation was observed. Moreover, in comparison with the control group, a reduced number of cells were observed. Based on these results, it considered that the postbiotic compounds from Lactobacillus La1 and La2 could provide crucial information in the development of anticancer research. Through further research, it would be beneficial to investigate the possibility of using these postbiotics (Pobt-La1 and -La2) as an anticancer drug.
In this work, solar-light-active copper–vanadium oxide (Cu-VO) was synthesized by a simple microwave method and characterized by FESEM, EDS, XRD, XPS, UV–Vis/near-infrared (NIR), and FT-IR spectroscopy. Antialgal and dye degradation activities of Cu-VO were investigated against Microcystis aeruginosa and methylene blue dye (MB), respectively. The mechanism of action of Cu-VO was examined regarding the production of hydroxyl radical (·OH) in the medium and intracellular reactive oxygen species (ROS) in M. aeruginosa. FESEM and XRD analyses of Cu-VO disclosed the formation of monoclinic crystals with an average diameter of 132 nm. EDX and XPS analyses showed the presence of Cu, V, and O atoms on the surface of Cu-VO. Furthermore, FT-IR analysis of Cu-VO exposed the presence of tetrahedral VO4 and octahedral CuO6. Cu-VO effectively reduced the algal growth and degraded methylene blue under solar light. A total of 4 mg/L of Cu-VO was found to be effective for antialgal activity. Cu-VO degraded 93% of MB. The investigation of the mechanism of action of Cu-VO showed that ·OH mediated antialgal and dye degradation of M. aeruginosa and MB. Cu-VO also triggered the production of intracellular ROS in M. aeruginosa, leading to cell death. Thus, Cu-VO could be an effective catalyst for wastewater treatment.
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