Structural and functional insights into the 2'-O-methyltransferase of SARS-CoV-2.

A unique feature of coronaviruses is their utilization of self-encoded nonstructural protein 16 (nsp16), 2'-O-methyltransferase (2'-O-MTase), to cap their RNAs through ribose 2'-O-methylation modification. This process is crucial for maintaining viral genome stability, facilitating efficient translation, and enabling immune escape. Despite considerable advances in the ultrastructure of SARS-CoV-2 nsp16/nsp10, insights into its molecular mechanism have so far been limited. In this study, we systematically characterized the 2'-O-MTase activity of nsp16 in SARS-CoV-2, focusing on its dependence on nsp10 stimulation. We observed cross-reactivity between nsp16 and nsp10 in various coronaviruses due to a conserved interaction interface. However, a single residue substitution (K58T) in SARS-CoV-2 nsp10 restricted the functional activation of MERS-CoV nsp16. Furthermore, the cofactor nsp10 effectively enhanced the binding of nsp16 to the substrate RNA and the methyl donor S-adenosyl-l-methionine (SAM). Mechanistically, His-80, Lys-93, and Gly-94 of nsp10 interacted with Asp-102, Ser-105, and Asp-106 of nsp16, respectively, thereby effectively stabilizing the SAM binding pocket. Lys-43 of nsp10 interacted with Lys-38 and Gly-39 of nsp16 to dynamically regulate the RNA binding pocket and facilitate precise binding of RNA to the nsp16/nsp10 complex. By assessing the conformational epitopes of nsp16/nsp10 complex, we further determined the critical residues involved in 2'-O-MTase activity. Additionally, we utilized an in vitro biochemical platform to screen potential inhibitors targeting 2'-O-MTase activity. Overall, our results significantly enhance the understanding of viral 2'-O methylation process and mechanism, providing valuable targets for antiviral drug development.

CD-Loop: a chromatin loop detection method based on the diffusion model.

Motivation: In recent years, there have been significant advances in various chromatin conformation capture techniques, and annotating the topological structure from Hi-C contact maps has become crucial for studying the three-dimensional structure of chromosomes. However, the structure and function of chromatin loops are highly dynamic and diverse, influenced by multiple factors. Therefore, obtaining the three-dimensional structure of the genome remains a challenging task. Among many chromatin loop prediction methods, it is difficult to fully extract features from the contact map and make accurate predictions at low sequencing depths. Results: In this study, we put forward a deep learning framework based on the diffusion model called CD-Loop for predicting accurate chromatin loops. First, by pre-training the input data, we obtain prior probabilities for predicting the classification of the Hi-C contact map. Then, by combining the denoising process based on the diffusion model and the prior probability obtained by pre-training, candidate loops were predicted from the input Hi-C contact map. Finally, CD-Loop uses a density-based clustering algorithm to cluster the candidate chromatin loops and predict the final chromatin loops. We compared CD-Loop with the currently popular methods, such as Peakachu, Chromosight, and Mustache, and found that in different cell types, species, and sequencing depths, CD-Loop outperforms other methods in loop annotation. We conclude that CD-Loop can accurately predict chromatin loops and reveal cell-type specificity. The code is available at https://github.com/wangyang199897/CD-Loop.

CineMol: a programmatically accessible direct-to-SVG 3D small molecule drawer.

Effective visualization of small molecules is paramount in conveying concepts and results in cheminformatics. Scalable vector graphics (SVG) are preferred for creating such visualizations, as SVGs can be easily altered in post-production and exported to other formats. A wide spectrum of software applications already exist that can visualize molecules, and customize these visualizations, in many ways. However, software packages that can output projected 3D models onto a 2D canvas directly as SVG, while being programmatically accessible from Python, are lacking. Here, we introduce CineMol, which can draw vectorized approximations of three-dimensional small molecule models in seconds, without triangulation or ray tracing, resulting in files of around 50-300 kilobytes per molecule model for compounds with up to 45 heavy atoms. The SVGs outputted by CineMol can be readily modified in popular vector graphics editing software applications. CineMol is written in Python and can be incorporated into any existing Python cheminformatics workflow, as it only depends on native Python libraries. CineMol also provides programmatic access to all its internal states, allowing for per-atom and per-bond-based customization. CineMol's capacity to programmatically create molecular visualizations suitable for post-production offers researchers and scientists a powerful tool for enhancing the clarity and visual impact of their scientific presentations and publications in cheminformatics, metabolomics, and related scientific disciplines.Scientific contributionWe introduce CineMol, a Python-based tool that provides a valuable solution for cheminformatics researchers by enabling the direct generation of high-quality approximations of two-dimensional SVG visualizations from three-dimensional small molecule models, all within a programmable Python framework. CineMol offers a unique combination of speed, efficiency, and accessibility, making it an indispensable tool for researchers in cheminformatics, especially when working with SVG visualizations.

Three-Dimensional Vanadium and Nitrogen Dual-Doped Ti3C2 Film with Ultra-High Specific Capacitance and High Volumetric Energy Density for Zinc-Ion Hybrid Capacitors.

Zinc-ion hybrid capacitors (ZICs) can achieve high energy and power density, ultralong cycle life, and a wide operating voltage window, and they are widely used in wearable devices, portable electronics devices, and other energy storage fields. The design of advanced ZICs with high specific capacity and energy density remains a challenge. In this work, a novel kind of V, N dual-doped Ti3C2 film with a three-dimensional (3D) porous structure (3D V-, N-Ti3C2) based on Zn-ion pre-intercalation can be fabricated via a simple synthetic process. The stable 3D structure and heteroatom doping provide abundant ion transport channels and numerous surface active sites. The prepared 3D V-, N-Ti3C2 film can deliver unexpectedly high specific capacitance of 855 F g-1 (309 mAh g-1) and demonstrates 95.26% capacitance retention after 5000 charge/discharge cycles. In addition, the energy storage mechanism of 3D V-, N-Ti3C2 electrodes is the chemical adsorption of H+/Zn2+, which is confirmed by ex situ XRD and ex situ XPS. ZIC full cells with a competitive energy density (103 Wh kg-1) consist of a 3D V-, N-Ti3C2 cathode and a zinc foil anode. The impressive results provide a feasible strategy for developing high-performance MXene-based energy storage devices in various energy-related fields.

Giant sequoia (Sequoiadendron giganteum) in the UK: carbon storage potential and growth rates.

Giant sequoias (Sequoiadendron giganteum) are some of the UK's largest trees, despite only being introduced in the mid-nineteenth century. There are an estimated half a million giant sequoias and closely related coastal redwoods (Sequoia sempervirens) in the UK. Given the recent interest in planting more trees, partly due to their carbon sequestration potential and also their undoubted public appeal, an understanding of their growth capability is important. However, little is known about their growth and carbon uptake under UK conditions. Here, we focus on S. giganteum and use three-dimensional terrestrial laser scanning to perform detailed structural measurements of 97 individuals at three sites covering a range of different conditions, to estimate aboveground biomass (AGB) and annual biomass accumulation rates. We show that UK-grown S. giganteum can sequester carbon at a rate of 85 kg yr-1, varying with climate, management and age. We develop new UK-specific allometric models for S. giganteum that fit the observed AGB with r 2 > 0.93 and bias < 2% and can be used to estimate S. giganteum biomass more generally. This study provides the first estimate of the growth and carbon sequestration of UK open-grown S. giganteum and provides a baseline for estimating their longer-term carbon sequestration capacity.

The utility of the psycholexical approach for identifying military core values: Illustrated in a sample of Swiss career officers and NCOs.

Values have always been a top priority in the military domains of leadership, training, ethical commitment, and psychological research. However, only a few studies have assessed value descriptors and their underlying dimensional structure in military organizations using an empirical psycholexical and factor analytical approach. This research project examined the structure of military values and derived core military values. Two studies were conducted in cooperation with the Swiss Armed Forces. In study 1, 25 military-specific value descriptors were identified based on a psycholexical analysis of military guidelines and in line with expert ratings by executive military leaders. In study 2, a questionnaire was filled out by a sample of 550 military professionals to capture their ratings of values as applied to everyday military decisions and actions. Principal component analysis in combination with Goldberg's top-down approach delivered five military value categories that reflect the military culture in Switzerland, characterized as (I) freedom, (II) social cohesion, (III) good soldiership, (IV) mutual respect, and (V) military conformity. Results are discussed in light of introducing a novel research approach to assessing the value structure and culture in military organizations.

Colossal Magnetoresistance in Layered Diluted Magnetic Semiconductor Rb(Zn,Li,Mn)4As3 Single Crystals.

Diluted magnetic semiconductors (DMSs) with tunable ferromagnetism are among the most promising materials for fabricating spintronic devices. Some DMS systems have sizeable magnetoresistances that can further extend their applications. Here, we report a new DMS Rb(Zn1-x-yLiyMnx)4As3 with a quasi-two-dimensional structure showing sizeable anisotropies in its ferromagnetism and transverse magnetoresistance (MR). With proper charge and spin doping, single crystals of the DMS display Curie temperatures up to 24 K. Analysis of the critical behavior via Arrott plots confirms the long-range ferromagnetic ordering in the Rb(Zn1-x-yLiyMnx)4As3 single crystals. We observed remarkable intrinsic MR effects in the single crystals (i.e., a positive MR of 85% at 0.4 T and a colossal negative MR of -93% at 7 T).

A review: analysis of technical challenges in cultured meat production and its commercialization.

The cultured meat technology has developed rapidly in recent years, but there are still many technical challenges that hinder the large-scale production and commercialization of cultured meat. Firstly, it is necessary to lay the foundation for cultured meat production by obtaining seed cells and maintaining stable cell functions. Next, technologies such as bioreactors are used to expand the scale of cell culture, and three-dimensional culture technologies such as scaffold culture or 3D printing are used to construct the three-dimensional structure of cultured meat. At the same time, it can reduce production costs by developing serum-free medium suitable for cultured meat. Finally, the edible quality of cultured meat is improved by evaluating food safety and sensory flavor, and combining ethical and consumer acceptability issues. Therefore, this review fully demonstrates the current development status and existing technical challenges of the cultured meat production technology with regard to the key points described above, in order to provide research ideas for the industrial production of cultured meat.

The reversible activation of norovirus by metal ions.

Murine norovirus (MNV) undergoes extremely large conformational changes in response to the environment. The T = 3 icosahedral capsid is composed of 180 copies of ~58-kDa VP1 comprised of N-terminus (N), shell (S), and C-terminal protruding (P) domains. At neutral pH, the P domains are loosely tethered to the shell and float ~15 Å above the surface. At low pH or in the presence of bile salts, the P domain drops onto the shell and this movement is accompanied by conformational changes within the P domain that enhance receptor interactions while blocking antibody binding. While previous crystallographic studies identified metal binding sites in the isolated P domain, the ~2.7-Å cryo-electron microscopy structures of MNV in the presence of Mg2+ or Ca2+ presented here show that metal ions can recapitulate the contraction observed at low pH or in the presence of bile. Further, we show that these conformational changes are reversed by dialysis against EDTA. As observed in the P domain crystal structures, metal ions bind to and contract the G'H' loop. This movement is correlated with the lifting of the C'D' loop and rotation of the P domain dimers about each other, exposing the bile salt binding pocket. Isothermal titration calorimetry experiments presented here demonstrate that the activation signals (bile salts, low pH, and metal ions) act in a synergistic manner that, individually, all result in the same activated structure. We present a model whereby these reversible conformational changes represent a uniquely dynamic and tissue-specific structural adaptation to the in vivo environment.IMPORTANCEThe highly mobile protruding domains on the calicivirus capsids are recognized by cell receptor(s) and antibodies. At neutral pH, they float ~15 Å above the shell but at low pH or in the presence of bile salts, they contract onto the surface. Concomitantly, changes within the P domain block antibody binding while enhancing receptor binding. While we previously demonstrated that metals also block antibody binding, it was unknown whether they might also cause similar conformational changes in the virion. Here, we present the near atomic cryo-electron microscopy structures of infectious murine norovirus (MNV) in the presence of calcium or magnesium ions. The metal ions reversibly induce the same P domain contraction as low pH and bile salts and act in a synergistic manner with the other stimuli. We propose that, unlike most other viruses, MNV facilely changes conformations as a unique means to escape immune surveillance as it moves through various tissues.

The Site/Group Extended Data Format and Tools.

Comparative sequence analysis permits unraveling the molecular processes underlying gene evolution. Many statistical methods generate candidate positions within genes, such as fast or slowly evolving sites, coevolving groups of residues, sites undergoing positive selection, or changes in evolutionary rates. Understanding the functional causes of these evolutionary patterns requires combining the results of these analyses and mapping them onto molecular structures, a complex task involving distinct coordinate referential systems. To ease this task, we introduce the site/group extended data format, a simple text format to store (groups of) site annotations. We developed a toolset, the SgedTools, which permits site/group extended data file manipulation, creating them from various software outputs and translating coordinates between individual sequences, alignments, and three-dimensional structures. The package also includes a Monte-Carlo procedure to generate random site samples, possibly conditioning on site-specific features. This eases the statistical testing of evolutionary hypotheses, accounting for the structural properties of the encoded molecules.

Heterogeneity of cancer stem cell-related marker expression is associated with three-dimensional structures in malignant pleural effusion produced by lung adenocarcinoma.

INTRODUCTION: Cancer stem cells have been described in lung adenocarcinoma-associated malignant pleural effusion. They show clinically important features, including the ability to initiate new tumours and resistance to treatments. However, their correlation with the three-dimensional tumour structures in the effusion is not well understood. METHODS: Cell blocks produced from lung adenocarcinoma patients' pleural effusion were examined for cancer stem cell-related markers Nanog and CD133 using immunocytochemistry. The three-dimensional cancer cell structures and CD133 expression patterns were visualized with tissue-clearing technology. The expression patterns were correlated with tumour cell structures, genetic variants and clinical outcomes. RESULTS: Thirty-nine patients were analysed. Moderate-to-strong Nanog expression was detected in 27 cases (69%), while CD133 was expressed by more than 1% of cancer cells in 11 cases (28%). Nanog expression was more homogenous within individual specimens, while CD133 expression was detected in single tumour cells or cells within small clusters instead of larger structures in 8 of the 11 positive cases (73%). Although no statistically significant correlation between the markers and tumour genetic variants or patient survival was observed, we recorded seven cases with follow-up specimens after cancer treatment, and four (57%) showed a change in stem cell-related marker expression corresponding to treatment response. CONCLUSIONS: Lung adenocarcinoma cells in the pleural effusion show variable expression of cancer stem cell-related markers, some showing a correlation with the size of cell clusters. Their expression level is potentially correlated with cancer treatment effects.

Progress of Dispersants for Coal Water Slurry.

Dispersants, serving as an essential raw material in the formulation of coal water slurry, offer an economical and convenient solution for enhancing slurry concentration, thus stimulating significant interest in the development of novel and efficient dispersants. This paper intends to illuminate the evolution of dispersants by examining both the traditional and the newly conceived types and elaborating on their respective mechanisms of influence on slurry performance. Dispersants can be classified into anionic, cationic, amphoteric, and non-ionic types based on their dissociation properties. They can be produced by modifying either natural or synthetic products. The molecular structure of a dispersant allows for further categorization into one-dimensional, two-dimensional, or three-dimensional structure dispersants. This document succinctly outlines dispersants derived from natural products, three-dimensional structure dispersants, common anionic dispersants such as lignin and naphthalene, and amphoteric and non-ionic dispersants. Subsequently, the adsorption mechanism of dispersants, governed by either electrostatic attraction or functional group effects, is elucidated. The three mechanisms through which dispersants alter the surface properties of coal, namely the wetting dispersion effect, electrostatic repulsion effect, and steric hindrance effect, are also explained. The paper concludes with an exploration of the challenges and emerging trends in the domain of dispersants.

Psychometric properties of the Polish version of the Post-traumatic Stress Disorder Check List according to DSM-5 - PCL-5. / Wlasciwosci psychometryczne polskiej wersji Listy Zaburzen po Stresie Traumatycznym wedlug DSM-5 ­ PCL-5.

OBJECTIVES: The changes in the structure of PTSD symptoms introduced in the DSM-5 classification require the use of an appropriate measurement tool. For this purpose, the PTSD Checklist (PCL-5) was constructed and popularized. In the presented studies, the psychometric properties of the Polish version of PCL-5 were assessed. In addition, referring to the controversy regarding the conceptualization of PTSD, various indicators of the fit of five PTSD structure models were checked based on our own research. METHODS: People (N = 1035) who experienced various traumatic events participated in the anonymous research. All completed PCL-5 and another questionnaires used to assess the validity of PCl-5. RESULTS: The psychometric properties of the Polish version of PCL-5 are satisfactory. In the differential diagnosis the optimal point of discrimination is the result of ⩾ 33. The CFA results showed that all tested PTSD models met the basic fit criteria. The four-factor model explained 58% of the variance, including changes in arousal and reactivity of 36%. CONCLUSIONS: PCL-5 is a reliable and accurate tool for PTSD measurement. It is used for the initial diagnosis of PTSD. The conducted analyzes, despite demonstrating the diagnostical utility of PCL-5, do not indicate ultimately favor a single PTSD dimensionality model.

SARS-CoV-2 proteins structural studies using synchrotron radiation.

In the process of the development of structural biology, both the size and the complexity of the determined macromolecular structures have grown significantly. As a result, the range of application areas for the results of structural studies of biological macromolecules has expanded. Significant progress in the development of structural biology methods has been largely achieved through the use of synchrotron radiation. Modern sources of synchrotron radiation allow to conduct high-performance structural studies with high temporal and spatial resolution. Thus, modern techniques make it possible to obtain not only static structures, but also to study dynamic processes, which play a key role in understanding biological mechanisms. One of the key directions in the development of structural research is the drug design based on the structures of biomolecules. Synchrotron radiation offers insights into the three-dimensional time-resolved structure of individual viral proteins and their complexes at atomic resolution. The rapid and accurate determination of protein structures is crucial for understanding viral pathogenicity and designing targeted therapeutics. Through the application of experimental techniques, including X-ray crystallography and small-angle X-ray scattering (SAXS), it is possible to elucidate the structural details of SARS-CoV-2 virion containing 4 structural, 16 nonstructural proteins (nsp), and several accessory proteins. The most studied potential targets for vaccines and drugs are the structural spike (S) protein, which is responsible for entering the host cell, as well as nonstructural proteins essential for replication and transcription, such as main protease (Mpro), papain-like protease (PLpro), and RNA-dependent RNA polymerase (RdRp). This article provides a brief overview of structural analysis techniques, with focus on synchrotron radiation-based methods applied to the analysis of SARS-CoV-2 proteins.

Identification and characterization of cold-responsive aquaporins from the larvae of a crambid pest Agriphila aeneociliella (Eversmann) (Lepidoptera: Crambidae).

As small ectotherms, insects need to cope with the challenges of winter cold by regulating the water content through water transport. Aquaporins (AQPs) are key players to enhance the cold resistance by mediating essential homeostatic processes in many animals but remain poorly characterized in insects. Agriphila aeneociliella is a newly discovered winter wheat pest in China, and its early-stage larvae have strong tolerance to low temperature stress. Six AQP genes were identified, which belong to five AQP subfamilies (RPIP, Eglp, AQP12L, PRIP, DRIP). All of them contained six hydrophobic transmembrane helices (TMHs) and two relatively conservative Asparagine-Proline-Alanine motifs. The three-dimensional homology modeling showed that the six TMHs folded into an hourglass-like shape, and the imperceptible replace of four ar/R residues in contraction region had critical effects on changing the pore size of channels. Moreover, the transcript levels of AaAQP 1, 3, and 6 increased significantly with the treatment time below 0 °C. Combined with the results of pore radius variation, it is suggested that AaAQP1 and AaAQP3 may be considered to be the key anti-hypothermia proteins in A. aeneociliella by regulating rapid cell dehydration and allowing the influx of extracellular cold resistance molecules, thus avoiding death in winter.

MpADC, an L-aspartate-α-decarboxylase, from Myzus persicae, that enables production of ß-alanine with high yield by whole-cell enzymatic catalysis.

BACKGROUND: ß-Alanine is a precursor of many important pharmaceutical products and food additives, its market demand is continuously increasing nowadays. Whole-cell catalysis relying on the recombinant expression of key ß-alanine synthesizing enzymes is an important method to produce ß-alanine. Nevertheless, ß-alanine synthesizing enzymes found so far have problems including easy inactivation, low expression or poor catalytic activity, and it remains necessary to develop new enzymes. RESULTS: Herein, we characterized an L-aspartate-α-decarboxylase, MpADC, from an aphid, Myzus persicae. It showed excellent catalytic activity at pH 6.0-7.5 and 37 °C. With the help of chaperone co-expression and N-terminal engineering guided by AlphaFold2 structure prediction, the expression and catalytic ability of MpADC in Escherichia coli were significantly improved. Using 50 g/L of E. coli cells expressing the MpADC-∆39 variant cultured in a 15-L fermenter, 232.36 g/L of ß-alanine was synthesized in 13.5 h, with the average ß-alanine yield of 17.22 g/L/h, which is best known so far. CONCLUSIONS: Our research should facilitate the production of ß-alanine in an environment-friendly manner.

Taking stock of the mutations in human SARS-CoV-2 spike proteins: From early days to nearly the end of COVID-19 pandemic.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), causative agent of the coronavirus disease-2019 (COVID-19) has resulted in several deaths and severe economic losses throughout the world. The spike protein in the virus binds to the human ACE-2 receptor in order to mediate virus-host interactions required for the viral transmission. Since first report of the SARS-CoV-2 sequence during December 2019 from patient infected with the virus in Wuhan, China, the virus has undergone rapid changes leading to mutations comprising substitutions, deletions and insertions in the sequence resulting in several variants of the virus that were more virulent and transmissible or less virulent but highly transmissible. The timely intervention with COVID-19 vaccines proved to be effective in controlling the number of infections. However, rapid mutations in the virus led to the lowering of vaccine efficacies being administered to people. In May 2023, the World Health Organization declared COVID-19 was not a public health emergency of international concern anymore. In order to take stock of mutations in the virus from early days to nearly end of COVID-19 pandemic, sequence analyses of the SARS-CoV-2 spike proteins available in the NCBI Virus database was carried out. The mutations and invariant residues in the SARS-CoV-2 spike protein sequences relative to the reference sequence were analysed. The location of the invariant residues and residues at interface of the protein chains in the spike protein trimer complex structure were examined. A total of 111,298 non-redundant SARS-CoV-2 spike protein sequences representing 2,345,585 spike proteins in the NCBI Virus database showed mutations at 1252 of the 1273 positions in the amino acid sequence. The mutations represented 6129 different mutation types in the sequences analysed. Besides, some sequences also contained insertion mutations. The SARS-CoV-2 spike protein sequences represented 1435 lineages. In addition, several spike protein sequences with mutations whose lineages were either 'not classified' or were 'unclassifiable' indicated the virus could still be evolving.

Structural and antigenic characterization of the avian adeno-associated virus capsid.

IMPORTANCE: AAVs are extensively studied as promising therapeutic gene delivery vectors. In order to circumvent pre-existing antibodies targeting primate-based AAV capsids, the AAAV capsid was evaluated as an alternative to primate-based therapeutic vectors. Despite the high sequence diversity, the AAAV capsid was found to bind to a common glycan receptor, terminal galactose, which is also utilized by other AAVs already being utilized in gene therapy trials. However, contrary to the initial hypothesis, AAAV was recognized by approximately 30% of human sera tested. Structural and sequence comparisons point to conserved epitopes in the fivefold region of the capsid as the reason determinant for the observed cross-reactivity.

Isolation and protein MdtQ analysis of outer membrane vesicles released by carbapenem-resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a leading public health problem, and is increasingly being reported worldwide with resistance to a wide spectrum of antibiotics. Recent reports have demonstrated that the outer membrane vesicles (OMVs) of gram-negative bacteria are potent resistance factors, but their role in the drug resistance of CRKP has not been elucidated. In order to investigate the effects of OMV components on drug resistance and to explore the mechanism of antimicrobial resistance in CRKP, we isolated the OMVs through ultracentrifugation, separated the OMV proteins through mass spectrometry (MS), and performed bioinformatics analysis. A total of 3,192 proteins were detected by nano LC-MS/MS analysis, with 108 (61.4%) cytoplasmic proteins, 50 (28.4%) cytoplasmic membrane proteins, nine (5.1%) periplasmic proteins, six (3.4%) outer membrane proteins, two (1.1%) extracellular proteins, and one (0.6%) other protein detected in the vesicles. MdtQ was detected as the only multidrug resistance outer membrane protein. Further experiments confirmed that MdtQ included the 1440 BP sequence and had a unique three-dimensional structure. To superimpose MdtQ with KPC-2 resistant proteins, I7ACB1, I7AKP2, and Q93LQ9, the root mean square deviation (RMSD) values were calculated (0.379, 0.671, and 1.35, respectively). I7ACB1 had the lowest RMSD value, indicating that it had the best superimposition effect. Furthermore, MdtQ had 20 biological pocket structures, and the four most important pockets were evenly distributed around the inner perimeter of its three-dimensional structure. These findings may provide a theoretical basis for controlling the spread of bacterial resistance in the future.

Functional significance of serine 13 in the active site of glutathione S-transferase F3 from Oryza sativa.

Plant glutathione S-transferase (GST, EC 2.5.1.18) is an enzyme that detoxifies various electrophilic compounds including herbicides and organic pollutants by catalyzing the formation of conjugates with reduced glutathione (GSH). Although the structure and function of the GST subunits in rice, an important food in Asia, are not well understood, they are crucial for herbicide development. To investigate the role of active site residues in rice Phi-class GSTF3 (OsGSTF3), evolutionarily conserved serine residues were replaced with alanine using site-directed mutagenesis to obtain the mutants S13A, S38A, S69A, and S169A. These four mutants were expressed in Escherichia coli and purified to electrophoretic homogeneity using immobilized GSH affinity chromatography. Mutation of Ser13 to Ala resulted in substantial reductions in specific activities and kcat/Km values for the GSH-[1-chloro-2,4-dinitrobenzene (CDNB)] conjugation reaction. In contrast, mutations of Ser38, Ser69, and Ser169 to Ala had little effect on the activities and kinetic parameters. Additionally, the mutation of Ser13 to Ala significantly affected the KmGSH and I50 values of S-hexylglutathione and S-(2,4-dinitrophenyl)glutathione, which compete with GSH and the product of GSH-CDNB conjugation, respectively. A pH-log (kcat/KmCDNB) plot was used to estimate the pKa value of GSH in the enzyme-GSH complex of the wild-type enzyme, which was approximately 6.9. However, the pKa value of GSH in the enzyme-GSH complex of the S13A mutant was approximately 8.7, which was about 1.8 pK units higher than that of the wild-type enzyme. OsGSTF3 was also crystallized for crystallographic study, and the structure analyses revealed that Ser13 is located in the active site and that its side chain is in close proximity to the thiol group of glutathione bound in the enzyme. Based on these substitution effects on kinetic parameters, the dependence of kinetic parameters on the pH and 3-dimensional structure, it was suggested that Ser13 in rice OsGSTF3 is the residue responsible for catalytic activity by lowering the pKa of GSH in the enzyme-GSH complex and enhancing the nucleophilicity of the GSH thiol in the active site.