The N-terminal peptide of the main protease of SARS-CoV-2, targeting dimer interface, inhibits its proteolytic activity.

The main protease (Mpro) of SARS-CoV-2 cleaves 11 sites of iral polypeptide chains and generates essential non-structural proteins for viral replication. Mpro is an important drug target against COVID-19. In this study, we developed a real-time fluorometric turn-on assay system to evaluate Mpro proteolytic activity for a substrate peptide between NSP4 and NSP5. It produced reproducible and reliable results suitable for HTS inhibitor assays. Thus far, most inhibitors against Mpro target the active site for substrate binding. Mpro exists as a dimer, which is essential for its activity. We investigated the potential of the Mpro dimer interface to act as a drug target. The dimer interface is formed of domain II and domain III of each protomer, in which N-terminal ten amino acids of the domain I are bound in the middle as a sandwich. The N-terminal part provides approximately 39% of the dimer interface between two protomers. In the real-time fluorometric turn-on assay system, peptides of the N-terminal ten amino acids, N10, can inhibit the Mpro activity. The dimer interface could be a prospective drug target against Mpro. The N-terminal sequence can help develop a potential inhibitor. [BMB Reports 2023; 56(11): 606-611].

Antiviral Activity Against SARS-CoV-2 Variants Using in Silico and in Vitro Approaches.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emergence in 2019 led to global health crises and the persistent risk of viral mutations. To combat SARS-CoV-2 variants, researchers have explored new approaches to identifying potential targets for coronaviruses. This study aimed to identify SARS-CoV-2 inhibitors using drug repurposing. In silico studies and network pharmacology were conducted to validate targets and coronavirus-associated diseases to select potential candidates, and in vitro assays were performed to evaluate the antiviral effects of the candidate drugs to elucidate the mechanisms of the viruses at the molecular level and determine the effective antiviral drugs for them. Plaque and cytopathic effect reduction were evaluated, and real-time quantitative reverse transcription was used to evaluate the antiviral activity of the candidate drugs against SARS-CoV-2 variants in vitro. Finally, a comparison was made between the molecular docking binding affinities of fenofibrate and remdesivir (positive control) to conventional and identified targets validated from protein-protein interaction (PPI). Seven candidate drugs were obtained based on the biological targets of the coronavirus, and potential targets were identified by constructing complex disease targets and PPI networks. Among the candidates, fenofibrate exhibited the strongest inhibition effect 1 h after Vero E6 cell infection with SARS-CoV-2 variants. This study identified potential targets for coronavirus disease (COVID-19) and SARS-CoV-2 and suggested fenofibrate as a potential therapy for COVID-19.

Structural Insights for ß-Lactam Antibiotics.

Antibiotic resistance has emerged as a global threat to modern healthcare systems and has nullified many commonly used antibiotics. ß-Lactam antibiotics are among the most successful and occupy approximately two-thirds of the prescription antibiotic market. They inhibit the synthesis of the peptidoglycan layer in the bacterial cell wall by mimicking the D-Ala-D-Ala in the pentapeptide crosslinking neighboring glycan chains. To date, various ß-lactam antibiotics have been developed to increase the spectrum of activity and evade drug resistance. This review emphasizes the three-dimensional structural characteristics of ß-lactam antibiotics regarding the overall scaffold, working mechanism, chemical diversity, and hydrolysis mechanism by ß-lactamases. The structural insight into various ß-lactams will provide an in-depth understanding of the antibacterial efficacy and susceptibility to drug resistance in multidrug-resistant bacteria and help to develop better ß-lactam antibiotics and inhibitors.

Heating-mediated purification of active FGF21 and structure-based design of its variant with enhanced potency.

Fibroblast growth factor 21 (FGF21) has pharmaceutical potential against obesity-related metabolic disorders, including non-alcoholic fatty liver disease. Since thermal stability is a desirable factor for therapeutic proteins, we investigated the thermal behavior of human FGF21. FGF21 remained soluble after heating; thus, we examined its temperature-induced structural changes using circular dichroism (CD). FGF21 showed inter-convertible temperature-specific CD spectra. The CD spectrum at 100 °C returned to that at 20 °C when the heated FGF21 solution was cooled. Through loop swapping, the connecting loop between ß10 and ß12 in FGF21 was revealed to be associated with the unique thermal behavior of FGF21. According to surface plasmon resonance (SPR) experiments, in vitro cell-based assays, and model high-fat diet (HFD)-induced obesity studies, heated FGF21 maintained biological activities that were comparable to those of non-heated and commercial FGF21s. Based on sequence comparison and structural analysis, five point-mutations were introduced into FGF21. Compared with the wild type, the heated FGF21 variant displayed improved therapeutic potential in terms of body weight loss, the levels of hepatic triglycerides and lipids, and the degree of vacuolization of liver in HFD-fed mice.

Transmission of antibiotic resistance genes through mobile genetic elements in Acinetobacter baumannii and gene-transfer prevention.

Antibiotic resistance is a major global public health concern. Acinetobacter baumannii is a nosocomial pathogen that has emerged as a global threat because of its high levels of resistance to many antibiotics, particularly those considered as last-resort antibiotics, such as carbapenems. Mobile genetic elements (MGEs) play an important role in the dissemination and expression of antibiotic resistance genes (ARGs), including the mobilization of ARGs within and between species. We conducted an in-depth, systematic investigation of the occurrence and dissemination of ARGs associated with MGEs in A. baumannii. We focused on a cross-sectoral approach that integrates humans, animals, and environments. Four strategies for the prevention of ARG dissemination through MGEs have been discussed: prevention of airborne transmission of ARGs using semi-permeable membrane-covered thermophilic composting; application of nanomaterials for the removal of emerging pollutants (antibiotics) and pathogens; tertiary treatment technologies for controlling ARGs and MGEs in wastewater treatment plants; and the removal of ARGs by advanced oxidation techniques. This review contemplates and evaluates the major drivers involved in the transmission of ARGs from the cross-sectoral perspective and ARG-transfer prevention processes.

Conformational change of organic cofactor PLP is essential for catalysis in PLP-dependent enzymes.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes are ubiquitous, catalyzing various biochemical reactions of approximately 4% of all classified enzymatic activities. They transform amines and amino acids into important metabolites or signaling molecules and are important drug targets in many diseases. In the crystal structures of PLP-dependent enzymes, organic cofactor PLP showed diverse conformations depending on the catalytic step. The conformational change of PLP is essential in the catalytic mechanism. In the study, we review the sophisticated catalytic mechanism of PLP, especially in transaldimination reactions. Most drugs targeting PLP-dependent enzymes make a covalent bond to PLP with the transaldimination reaction. A detailed understanding of organic cofactor PLP will help develop a new drug against PLP-dependent enzymes. [BMB Reports 2022; 55(9): 439-446].

Structural characterization and fatty acid epoxidation of CYP184A1 from Streptomyces avermitilis.

The genome of Streptomyces avermitilis contains 33 cytochrome P450 genes. Among the P450 gene products of S. avermitilis, we characterized the biochemical function and structural aspects of CYP184A1. Ultra-performance liquid chromatography-tandem mass spectrometry analysis showed that CYP184A1 induced an epoxidation reaction to produce 9,10-epoxystearic acid. Steady-state kinetic analysis yielded a kcat value of 0.0067 min-1 and a Km value 10 µM. The analysis of its crystal structures illustrated that the overall CYP184A1 structure adopts the canonical scaffold of cytochrome P450 and possesses a narrow and deep substrate pocket architecture that is required for binding to linear chain fatty acids. In the structure of the CYP184A1 oleic acid complex (CYP184A1-OA), C9-C10 of oleic acid was bound to heme for the productive epoxidation reaction. This study elucidates the roles of P450 enzymes in the oxidative metabolism of fatty acids in Streptomyces species.

Elucidating the Effects of Curcumin against Influenza Using In Silico and In Vitro Approaches.

The influenza virus is a constantly evolving pathogen that challenges medical and public health systems. Traditionally, curcumin has been used to treat airway inflammatory diseases, such as bronchitis and pneumonia. To elucidate common targets of curcumin and influenza infection and underlying mechanisms, we employed network pharmacology and molecular docking approaches and confirmed results using in vitro experiments. Biological targets of curcumin and influenza were collected, and potential targets were identified by constructing compound-disease target (C-D) and protein-protein interaction (PPI) networks. The ligand-target interaction was determined using the molecular docking method, and in vitro antiviral experiments and target confirmation were conducted to evaluate curcumin's effects on influenza. Our network and pathway analyses implicated the four targets of AKT1, RELA, MAPK1, and TP53 that could be involved in the inhibitory effects of curcumin on influenza. The binding energy calculations of each ligand-target interaction in the molecular docking showed that curcumin bound to AKT1 with the highest affinity among the four targets. In vitro experiments, in which influenza virus-infected MDCK cells were pre-, co-, or post-treated with curcumin, confirmed curcumin's prophylactic and therapeutic effects. Influenza virus induction increased the level of mRNA expression of AKT in MDCK cells, and the level was attenuated by curcumin treatment. Collectively, our findings identified potential targets of curcumin against influenza and suggest curcumin as a potential therapy for influenza infection.

Gold nanoparticles and tilt pairs to assess protein flexibility by cryo-electron microscopy.

A computational method was developed to recover the three-dimensional coordinates of gold nanoparticles specifically attached to a protein complex from tilt-pair images collected by electron microscopy. The program was tested on a simulated dataset and applied to a real dataset comprising tilt-pair images recorded by cryo electron microscopy of RNA polymerase II in a complex with four gold-labeled single-chain antibody fragments. The positions of the gold nanoparticles were determined, and comparison of the coordinates among the tetrameric particles revealed the range of motion within the protein complexes.

Combined Analysis of the Time-Resolved Transcriptome and Proteome of Plant Pathogen Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) is a plant pathogen responsible for causing bacterial blight in rice. The immediate alterations in Xoo upon initial contact with rice are essential for pathogenesis. We studied time-resolved genome-wide gene expression in pathogenicity-activated Xoo cells at the transcriptome and proteome levels. The early response genes of Xoo include genes related to cell motility, inorganic ion transport, and effectors. The alteration of gene expression is initiated as early as few minutes after the initial interaction and changes with time. The time-resolved comparison of the transcriptome and proteome shows the differences between transcriptional and translational expression peaks in many genes, although the overall expression pattern of mRNAs and proteins is conserved. The discrepancy suggests an important role of translational regulation in Xoo at the early stages of pathogenesis. The gene expression analysis using time-resolved transcriptome and proteome provides unprecedented valuable information regarding Xoo pathogenesis.

Structural Insights for Core Scaffold and Substrate Specificity of B1, B2, and B3 Metallo-ß-Lactamases.

Metallo-ß-lactamases (MBLs) hydrolyze almost all ß-lactam antibiotics, including penicillins, cephalosporins, and carbapenems; however, no effective inhibitors are currently clinically available. MBLs are classified into three subclasses: B1, B2, and B3. Although the amino acid sequences of MBLs are varied, their overall scaffold is well conserved. In this study, we systematically studied the primary sequences and crystal structures of all subclasses of MBLs, especially the core scaffold, the zinc-coordinating residues in the active site, and the substrate-binding pocket. We presented the conserved structural features of MBLs in the same subclass and the characteristics of MBLs of each subclass. The catalytic zinc ions are bound with four loops from the two central ß-sheets in the conserved αß/ßα sandwich fold of MBLs. The three external loops cover the zinc site(s) from the outside and simultaneously form a substrate-binding pocket. In the overall structure, B1 and B2 MBLs are more closely related to each other than they are to B3 MBLs. However, B1 and B3 MBLs have two zinc ions in the active site, while B2 MBLs have one. The substrate-binding pocket is different among all three subclasses, which is especially important for substrate specificity and drug resistance. Thus far, various classes of ß-lactam antibiotics have been developed to have modified ring structures and substituted R groups. Currently available structures of ß-lactam-bound MBLs show that the binding of ß-lactams is well conserved according to the overall chemical structure in the substrate-binding pocket. Besides ß-lactam substrates, B1 and cross-class MBL inhibitors also have distinguished differences in the chemical structure, which fit well to the substrate-binding pocket of MBLs within their inhibitory spectrum. The systematic structural comparison among B1, B2, and B3 MBLs provides in-depth insight into their substrate specificity, which will be useful for developing a clinical inhibitor targeting MBLs.

The Progression of SARS Coronavirus 2 (SARS-CoV2): Mutation in the Receptor Binding Domain of Spike Gene.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is a positive-sense single-stranded RNA (+ssRNA) that causes coronavirus disease 2019 (COVID-19). The viral genome encodes twelve genes for viral replication and infection. The third open reading frame is the spike (S) gene that encodes for the spike glycoprotein interacting with specific cell surface receptor - angiotensin converting enzyme 2 (ACE2) - on the host cell membrane. Most recent studies identified a single point mutation in S gene. A single point mutation in S gene leading to an amino acid substitution at codon 614 from an aspartic acid 614 into glycine (D614G) resulted in greater infectivity compared to the wild type SARS-CoV2. We were interested in investigating the mutation region of S gene of SARS-CoV2 from Korean COVID-19 patients. New mutation sites were found in the critical receptor binding domain (RBD) of S gene, which is adjacent to the aforementioned D614G mutation residue. This specific sequence data demonstrated the active progression of SARS-CoV2 by mutations in the RBD of S gene. The sequence information of new mutations is critical to the development of recombinant SARS-CoV2 spike antigens, which may be required to improve and advance the strategy against a wide range of possible SARS-CoV2 mutations.

Fructuronate-tagaturonate epimerase UxaE from Cohnella laeviribosi has a versatile TIM-barrel scaffold suitable for a sugar metabolizing biocatalyst.

Xylan and pectin are major structural components of plant cell walls. There are two independent catabolic pathways for xylan and pectin. UxaE bridges these two pathways by reversibly epimerizing D-fructuronate and D-tagaturonate. The crystal structure of UxaE from Cohnella laeviribosi (ClUxaE) shows a core scaffold of TIM-barrel with a position-changing divalent metal cofactor. ClUxaE has the flexible metal-coordination loop to allow the metal shift and the extra domains to bind a phosphate ion in the active site, which are important for catalysis and substrate specificity. Elucidation of the structure and mechanism of ClUxaE will assist in understanding the catalytic mechanism of UxaE family members, which are useful for processing both xylan and pectin-derived carbohydrates for practical and industrial purposes, including the transformation of agricultural wastes into numerous valuable products.

Structural insights into CYP107G1 from rapamycin-producing Streptomyces rapamycinicus.

Rapamycin is a clinically important macrolide agent with immunosuppressant and antiproliferative properties, produced by the actinobacterium, Streptomyces rapamycinicus. Two cytochrome P450 enzymes are involved in the biosynthesis of rapamycin. CYP107G1 and CYP122A2 catalyze the oxidation reactions of C27 and C9 of pre-rapamycin, respectively. To understand the structural and biochemical features of P450 enzymes in rapamycin biosynthesis, the CYP107G1 and CYP122A2 genes were cloned, their recombinant proteins were expressed in Escherichia coli, and the purified enzymes were characterized. Both enzymes displayed low spin states in the absolute spectra of ferric forms, and the titrations with rapamycin induced type I spectral changes with Kd values of 4.4 ± 0.4 and 3.0 ± 0.3 µM for CYP107G1 and CYP122A2, respectively. The X-ray crystal structures of CYP107G1 and its co-crystal complex with everolimus, a clinical rapamycin derivative, were determined at resolutions of 2.9 and 3.0 Å, respectively. The overall structure of CYP107G1 adopts the canonical scaffold of cytochrome P450 and possesses large substrate pocket. The distal face of the heme group is exposed to solvents to accommodate macrolide access. When the structure of the everolimus-bound CYP107G1 complex (CYP107G1-Eve) was compared to that of the ligand-free CYP107G1 form, no significant conformational change was observed. Hence, CYP107G1 has a relatively rigid structure with versatile loops to accommodate a bulky substrate. The everolimus molecule is bound to the substrate-binding pocket in the shape of a squeezed donut, and its elongated structure is bound perpendicular to a planar heme plane and I-helix.

Structural Study of Metal Binding and Coordination in Ancient Metallo-ß-Lactamase PNGM-1 Variants.

The increasing incidence of community- and hospital-acquired infections with multidrug-resistant (MDR) bacteria poses a critical threat to public health and the healthcare system. Although ß-lactam antibiotics are effective against most bacterial infections, some bacteria are resistant to ß-lactam antibiotics by producing ß-lactamases. Among ß-lactamases, metallo-ß-lactamases (MBLs) are especially worrisome as only a few inhibitors have been developed against them. In MBLs, the metal ions play an important role as they coordinate a catalytic water molecule that hydrolyzes ß-lactam rings. We determined the crystal structures of different variants of PNGM-1, an ancient MBL with additional tRNase Z activity. The variants were generated by site-directed mutagenesis targeting metal-coordinating residues. In PNGM-1, both zinc ions are coordinated by six coordination partners in an octahedral geometry, and the zinc-centered octahedrons share a common face. Structures of the PNGM-1 variants confirm that the substitution of a metal-coordinating residue causes the loss of metal binding and ß-lactamase activity. Compared with PNGM-1, subclass B3 MBLs lack one metal-coordinating residue, leading to a shift in the metal-coordination geometry from an octahedral to tetrahedral geometry. Our results imply that a subtle change in the metal-binding site of MBLs can markedly change their metal-coordination geometry and catalytic activity.

DNA Markers to Discriminate Cannabis sativa L. 'Cheungsam' with Low Tetrahydrocannabinol (THC) Content from Other South Korea Cultivars Based on the Nucleotide Sequences of Tetrahydrocannabinolic Acid Synthase and Putative 3-Ketoacyl-CoA Synthase Genes.

Cannabis sativa L. has been utilized for a long time as a traditional herbal medicine in Korea. Dry fruits, achenes, each containing a single seed of Cannabis, are currently prescribed as Ma In (Cannabis Semen), a laxative. As each achene is enclosed by a bract, in which tetrahydrocannabinol (THC), the main psychological active compound in Cannabis is synthesized; achene is easily contaminated by THC from bract remnants. Therefore, it is safer to harvest achenes from Cannabis with a low THC content. Seeds of hemp, a low THC Cannabis, were recently classified as possible sources of new pharmacologically active compounds. Thus, a proper method to select appropriate Cannabis plants with low THC among cultivars in South Korea for medicinal purpose is necessary. As a result of cross-selection, Cannabis L. cultivar "Cheungsam" (CH) with the lowest THC content among cultivars cultivated in South Korea has been developed. In this study, we developed two DNA markers to reliably discriminate CH from other local cultivars with higher THC contents. We developed primer sets CHF3/CHR2 to amplify the 642 bp DNA marker of CH based on differences in the nucleotide sequences of the THCA synthase gene, which encodes a key enzyme in THC synthesis. We then developed a CHF1/CHR3 primer set to amplify the 401 bp DNA marker of CH based on the differences in both the content of very long chain fatty acids (VLCFs) and the sequence of the putative 3-ketoacyl-CoA synthase (KCS) gene encoding enzymes synthesizing VLCFs among local cultivars.

Dual activity of PNGM-1 pinpoints the evolutionary origin of subclass B3 metallo-ß-lactamases: a molecular and evolutionary study.

Resistance to ß-lactams is one of the most serious problems associated with Gram-negative infections. ß-Lactamases are able to hydrolyze ß-lactams such as cephalosporins and/or carbapenems. Evolutionary origin of metallo-ß-lactamases (MBLs), conferring critical antibiotic resistance threats, remains unknown. We discovered PNGM-1, the novel subclass B3 MBL, in deep-sea sediments that predate the antibiotic era. Here, our phylogenetic analysis suggests that PNGM-1 yields insights into the evolutionary origin of subclass B3 MBLs. We reveal the structural similarities between tRNase Zs and PNGM-1, and demonstrate that PNGM-1 has both MBL and tRNase Z activities, suggesting that PNGM-1 is thought to have evolved from a tRNase Z. We also show kinetic and structural comparisons between PNGM-1 and other proteins including subclass B3 MBLs and tRNase Zs. These comparisons revealed that the B3 MBL activity of PNGM-1 is a promiscuous activity and subclass B3 MBLs are thought to have evolved through PNGM-1 activity.

The novel metallo-ß-lactamase PNGM-1 from a deep-sea sediment metagenome: crystallization and X-ray crystallographic analysis.

Metallo-ß-lactamases (MBLs) are present in major Gram-negative pathogens and environmental species, and pose great health risks because of their ability to hydrolyze the ß-lactam rings of antibiotics such as carbapenems. PNGM-1 was the first reported case of a subclass B3 MBL protein that was identified from a metagenomic library from deep-sea sediments that predate the antibiotic era. In this study, PNGM-1 was overexpressed, purified and crystallized. Crystals of native and selenomethionine-substituted PNGM-1 diffracted to 2.10 and 2.30 Šresolution, respectively. Both the native and the selenomethionine-labelled PNGM-1 crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 122, b = 83, c = 163 Å, ß = 110°. Matthews coefficient (VM) calculations suggested the presence of 6-10 molecules in the asymmetric unit, corresponding to a solvent content of ∼31-58%. Structure determination is currently in progress.

Crystal structures of an atypical aldehyde dehydrogenase having bidirectional oxidizing and reducing activities.

Aldehyde dehydrogenases (ALDHs) are NAD(P)+-dependent oxidoreductases that catalyze the oxidation of a variety of aldehydes to their acid forms. In this study, we determined the crystal structures of ALDH from Bacillus cereus (BcALDH), alone, and in complex with NAD+ and NADP+. This enzyme can oxidize all-trans-retinal to all-trans-retinoic acid using either NAD+ or NADP+ with equal efficiency, and atypically, as a minor activity, can reduce all-trans-retinal to all-trans-retinol using NADPH. BcALDH accommodated the additional 2'-phosphate of NADP+ by expanding the cofactor-binding pocket and upshifting the AMP moiety in NADP+. The nicotinamide moiety in NAD+ and NADP+ had direct interactions with the conserved catalytic residues (Cys300 and Glu266) and caused concerted conformational changes. We superimposed the structure of retinoic acid bound to human ALDH1A3 onto the BcALDH structure and speculated a model of the substrate all-trans-retinal bound to BcALDH. We also proposed a plausible mechanism for the minor reducing activity of BcALDH. These BcALDH structures will be useful in understanding cofactor specificity and the catalytic mechanism of an atypical bacterial BcALDH and should help the development of a new biocatalyst to produce retinoic acid and related high-end products.

Structure-based prediction and identification of 4-epimerization activity of phosphate sugars in class II aldolases.

Sugar 4-epimerization reactions are important for the production of rare sugars and their derivatives, which have various potential industrial applications. For example, the production of tagatose, a functional sweetener, from fructose by sugar 4-epimerization is currently constrained because a fructose 4-epimerase does not exist in nature. We found that class II D-fructose-1,6-bisphosphate aldolase (FbaA) catalyzed the 4-epimerization of D-fructose-6-phosphate (F6P) to D-tagatose-6-phosphate (T6P) based on the prediction via structural comparisons with epimerase and molecular docking and the identification of the condensed products of C3 sugars. In vivo, the 4-epimerization activity of FbaA is normally repressed. This can be explained by our results showing the catalytic efficiency of D-fructose-6-phosphate kinase for F6P phosphorylation was significantly higher than that of FbaA for F6P epimerization. Here, we identified the epimerization reactions and the responsible catalytic residues through observation of the reactions of FbaA and L-rhamnulose-1-phosphate aldolases (RhaD) variants with substituted catalytic residues using different substrates. Moreover, we obtained detailed potential epimerization reaction mechanism of FbaA and a general epimerization mechanism of the class II aldolases L-fuculose-1-phosphate aldolase, RhaD, and FbaA. Thus, class II aldolases can be used as 4-epimerases for the stereo-selective synthesis of valuable carbohydrates.