Active Site Domain Research Articles

Gibberellin-biosynthetic ent-kaurene synthases in higher plants do not require their non-catalytic domains for the catalysis.

ent-Kaurene is a biosynthetic intermediate diterpene of phytohormone gibberellins, and is biosynthesized from geranylgeranyl diphosphate via ent-copalyl diphosphate (ent-CDP). The successive cyclization is catalyzed by two distinct diterpene synthases, ent-CDP synthase (ent-CPS) and ent-kaurene synthase (KS). Homologs of these diterpene synthase genes have been reported to be involved in the biosynthesis of specialized-metabolic diterpenoids for defense in several plant species, including rice (Oryza sativa). These diterpene synthases consist of three domains, αβγ domains. Active sites of ent-CPS exist at the interface of β and γ domain, while those of KS are located within the α domain. We herein carried out domain-deletion experiments using several KSs and KS like enzymes (KSLs) to obtain insights into the roles of domains other than active-site domains. As previously reported in taxadiene synthase, deletion of γ or βγ domains drastically decreased activities of specialized-metabolic OsKSL5, OsKSL8, OsKSL7 and OsKSL10 in O. sativa. However, unexpectedly, only α domains of several gibberellin-biosynthetic KSs, including OsKS1 in O. sativa, AtKS in Arabidopsis thaliana, TaKS in wheat (Triticum aestivum) and BdKS1 in Brachypodium distachyon, retained their original functions. Additionally, the specialized-metabolic OsKSL4, which is closely related to OsKS1, also functioned without its βγ domains. Domain-swapping experiments showed that replacing βγ domains in OsKSL7 with those from other KS/KSLs retained the OsKSL7 activity. Moreover, deletion of βγ domains of bifunctional PpCPS/KS in moss (Physcomitrella patens) drastically impaired its KS-related activity. Thus, we demonstrate that monofunctional gibberellin-biosynthetic KSs are the unique diterpene synthases that retain their functions without βγ domains.

Architecture and regulation of filamentous human cystathionine beta-synthase

Cystathionine beta-synthase (CBS) is an essential metabolic enzyme across all domains of life for the production of glutathione, cysteine, and hydrogen sulfide. Appended to the conserved catalytic domain of human CBS is a regulatory domain that modulates activity by S-adenosyl-L-methionine (SAM) and promotes oligomerisation. Here we show using cryo-electron microscopy that full-length human CBS in the basal and SAM-bound activated states polymerises as filaments mediated by a conserved regulatory domain loop. In the basal state, CBS regulatory domains sterically block the catalytic domain active site, resulting in a low-activity filament with three CBS dimers per turn. This steric block is removed when in the activated state, one SAM molecule binds to the regulatory domain, forming a high-activity filament with two CBS dimers per turn. These large conformational changes result in a central filament of SAM-stabilised regulatory domains at the core, decorated with highly flexible catalytic domains. Polymerisation stabilises CBS and reduces thermal denaturation. In PC-3 cells, we observed nutrient-responsive CBS filamentation that disassembles when methionine is depleted and reversed in the presence of SAM. Together our findings extend our understanding of CBS enzyme regulation, and open new avenues for investigating the pathogenic mechanism and therapeutic opportunities for CBS-associated disorders.

The impact of pumpkin seed-derived silver nanoparticles on corrosion and cytotoxicity: a molecular docking study of the simulated AgNPs

ABSTRACT Green-synthesized nanoparticles from pumpkin (Cucurbita pepo L.) seed extracts are economical and eco-friendly. Silver nanoparticles (AgNPs) and their selective cytotoxicity towards HCT116 and African Green Monkey Kidney, Vero cells were investigated. Chemical fingerprinting, heat stability, and 2D-images of nanoparticle size and morphology were determined using Fourier-transform infrared spectroscopy (FTIR), Thermogravimetric Analysis (TGA), Transmission Electron Microscopy (TEM), and Scanning electron microscopy-energy dispersive X-ray (SEM-EDX) on AgNPs. UV-vis examination shows surface plasmons in the wide peak at 417 nm, indicating polydisperse nanoparticles. Small silver nanoparticles (AgNPs) below 2 µm demonstrated a rod-like form and a tendency to agglomerate. SEM-EDX element analysis and fingerprinting confirmed the material as AgNPs. TEM indicated that the nanoparticles were generally spherical or ellipsoidal, equally dispersed, and averaged 26.08 nm in diameter with negligible aggregation. The AgNPs are also stable at a temperature of 220°C, indicating that the green material is quite robust at 150°C to 400°C. According to cytotoxic studies, AgNPs are toxic to cancer cells (HCT 116 cells), however they have no effect on Vero cells. AgNPs and tubulin (TUB) domain active sites have a significant affinity, according to molecular docking analysis. In an electrochemical investigation, biogenic AgNPs effectively prevented mild steel from corroding in a 1.0 M HCl solution.

The Mechanism of Inhibition of Mycobacterial (p)ppGpp Synthetases by a Synthetic Analog of Erogorgiaene.

The synthesis of (p)ppGpp alarmones plays a vital role in the regulation of metabolism suppression, growth rate control, virulence, bacterial persistence, and biofilm formation. The (p)ppGpp alarmones are synthesized by proteins of the RelA/SpoT homolog (RSH) superfamily, including long bifunctional RSH proteins and small alarmone synthetases. Here, we investigated enzyme kinetics and dose-dependent enzyme inhibition to elucidate the mechanism of 4-(4,7-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)pentanoic acid (DMNP) action on the (p)ppGpp synthetases RelMsm and RelZ from Mycolicibacterium smegmatis and RelMtb from Mycobacterium tuberculosis. DMNP was found to inhibit the activity of RelMtb. According to the enzyme kinetics analysis, DMNP acts as a noncompetitive inhibitor of RelMsm and RelZ. Based on the results of molecular docking, the DMNP-binding site is located in the proximity of the synthetase domain active site. This study might help in the development of alarmone synthetase inhibitors, which includes relacin and its derivatives, as well as DMNP - a synthetic analog of the marine coral metabolite erogorgiaene. Unlike conventional antibiotics, alarmone synthetase inhibitors target metabolic pathways linked to the bacterial stringent response. Although these pathways are not essential for bacteria, they regulate the development of adaptation mechanisms. Combining conventional antibiotics that target actively growing cells with compounds that impede bacterial adaptation may address challenges associated with antimicrobial resistance and bacterial persistence.

Probing the Therapeutic Efficacy of Combretum paniculatum Extract and GC-FID-Identified Phytochemicals as Novel Agents for Diabetes Mellitus.

Oxidative stress is implicated in several metabolic cascades involved in glucose control. Hence, investigating antioxidant and antidiabetic activities is crucial for discovering an effective diabetes mellitus (DM) agent. This study was aimed at probing the therapeutic efficacy of hydro-ethanolic extract of Combretum paniculatum (HECP) and gas chromatography-flame ionization detector (GC-FID)-identified phytochemicals as novel agents for DM. We determined the total phenols, flavonoids, and antioxidant vitamins in HECP using standard methods. A GC-FID was used to decipher phytochemicals of HECP. The antioxidant and antidiabetic activities of HECP were assessed using in vitro and in silico approaches. The results revealed that HECP is affluent in phenols, flavonoids, and vitamin E and demonstrated engaging antioxidant activities in 1,1-diphenyl-2-picryl-hydroxyl (DPPH; IC50 = 0.83 µg/mL), thiobarbituric acid-reactive substances TBARS; IC50 = 2.28 µg/mL), and ferric-reducing antioxidant power assay (FRAP; IC50 = 2.89 µg/mL). Compared with the reference drug, acarbose, HECP exhibited good α-amylase and α-glucosidase inhibitory capacity, having IC50 values of 14.21 and 13.23 µg/mL, respectively, against 13.06 and 11.71 µg/mL recorded for acarbose. More so, the extract's top 6 scoring phytochemicals (rutin, kaempferol, epicatechin, ephedrine, naringenin, and resveratrol) had strong interactions with amino acid residues within and around α-amylase and α-glucosidase active site domains. All the compounds but rutin had favourable drug-like characteristics, pharmacokinetics, and safety profiles when compared with acarbose. Altogether, our results vindicate the use of this herb in treating DM locally and reveal that it has pharmaceutically active components that could be used as novel leads in the development of DM drugs.

An Integrated In Silico and In Vitro Approach for the Identification of Natural Products Active against SARS-CoV-2.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has provoked a global health crisis due to the absence of a specific therapeutic agent. 3CLpro (also known as the main protease or Mpro) and PLpro are chymotrypsin-like proteases encoded by the SARS-CoV-2 genome, and play essential roles during the virus lifecycle. Therefore, they are recognized as a prospective therapeutic target in drug discovery against SARS-CoV-2 infection. Thus, this work aims to collectively present potential natural 3CLpro and PLpro inhibitors by in silico simulations and in vitro entry pseudotype-entry models. We screened luteolin-7-O-glucuronide (L7OG), cynarin (CY), folic acid (FA), and rosmarinic acid (RA) molecules against PLpro and 3CLpro through a luminogenic substrate assay. We only reported moderate inhibitory activity on the recombinant 3CLpro and PLpro by L7OG and FA. Afterward, the entry inhibitory activity of L7OG and FA was tested in cell lines transduced with the two different SARS-CoV-2 pseudotypes harboring alpha (α) and omicron (o) spike (S) protein. The results showed that both compounds have a consistent inhibitory activity on the entry for both variants. However, L7OG showed a greater degree of entry inhibition against α-SARS-CoV-2. Molecular modeling studies were used to determine the inhibitory mechanism of the candidate molecules by focusing on their interactions with residues recognized by the protease active site and receptor-binding domain (RBD) of spike SARS-CoV-2. This work allowed us to identify the binding sites of FA and L7OG within the RBD domain in the alpha and omicron variants, demonstrating how FA is active in both variants. We have confidence that future in vivo studies testing the safety and effectiveness of these natural compounds are warranted, given that they are effective against a variant of concerns.

Natural Products as Potential Inhibitors of FLT3 for Acute Myeloid Leukemia: HTVS, Docking, and Molecular Dynamic Simulation

Cancer is one the most common health issues worldwide, with cancer-related mortality of 9.5 million in 2018, with an expectation to become 29.5 by 2040. Among others, acute myeloid leukemia (AML) is common among older people. FLT3 mutations are one of the most common genetic aberrations found in Acute Myeloid Leukemia and are associated with poor prognosis. Herein, we attempt to identify natural compounds as potential candidates to treat AML by targeting the FLT3 kinase domain using in silico approaches. The COCONUT database, which contains 407,270 natural compounds, was HTVS against the FLT3 kinase domain active site, and promising compounds were subject to molecular docking. Finally, frontier compounds were validated further using molecular dynamic simulation. In total, ten compounds were identified with docking scores higher than Quizartinib (-11.606 kcal/mol), with the best three compounds showing a docking score of -18.052, -15.772, and -16.767 kcal, respectively, and compound 2 showing excellent stability in molecular dynamic simulation.

Domain collapse and active site ablation generate a widespread animal mitochondrial seryl-tRNA synthetase.

Through their aminoacylation reactions, aminoacyl tRNA-synthetases (aaRS) establish the rules of the genetic code throughout all of nature. During their long evolution in eukaryotes, additional domains and splice variants were added to what is commonly a homodimeric or monomeric structure. These changes confer orthogonal functions in cellular activities that have recently been uncovered. An unusual exception to the familiar architecture of aaRSs is the heterodimeric metazoan mitochondrial SerRS. In contrast to domain additions or alternative splicing, here we show that heterodimeric metazoan mitochondrial SerRS arose from its homodimeric ancestor not by domain additions, but rather by collapse of an entire domain (in one subunit) and an active site ablation (in the other). The collapse/ablation retains aminoacylation activity while creating a new surface, which is necessary for its orthogonal function. The results highlight a new paradigm for repurposing a member of the ancient tRNA synthetase family.

NTRC regulates CP12 to activate Calvin–Benson cycle during cold acclimation

NADPH-dependent thioredoxin reductase C (NTRC) is a chloroplast redox regulator in algae and plants. Here, we used site-specific mutation analyses of the thioredoxin domain active site of NTRC in the green alga Chlamydomonas reinhardtii to show that NTRC mediates cold tolerance in a redox-dependent manner. By means of coimmunoprecipitation and mass spectrometry, a redox- and cold-dependent binding of the Calvin-Benson Cycle Protein 12 (CP12) to NTRC was identified. NTRC was subsequently demonstrated to directly reduce CP12 of C. reinhardtii as well as that of the vascular plant Arabidopsis thaliana in vitro. As a scaffold protein, CP12 joins the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to form an autoinhibitory supracomplex. Using size-exclusion chromatography, NTRC from both organisms was shown to control the integrity of this complex in vitro and thereby PRK and GAPDH activities in the cold. Thus, NTRC apparently reduces CP12, hence triggering the dissociation of the PRK/CP12/GAPDH complex in the cold. Like the ntrc::aphVIII mutant, CRISPR-based cp12::emx1 mutants also exhibited a redox-dependent cold phenotype. In addition, CP12 deletion resulted in robust decreases in both PRK and GAPDH protein levels implying a protein protection effect of CP12. Both CP12 functions are critical for preparing a repertoire of enzymes for rapid activation in response to environmental changes. This provides a crucial mechanism for cold acclimation.

An Insight into the Active sites of the RNA Polymerase and Proofreading Exonuclease of the Human Respiratory Syncytial Virus

Human respiratory syncytial virus (hRSV) is one of the triple epidemic viruses that causes infections of the respiratory tract and lungs. For multiplication of the virus in human cells, its RNA polymerase is the crucial enzyme and it forms a part of a large protein (LP). The LP is a multicomponent and multifunctional protein harboring at least 3 different enzymes. The RNA polymerase belongs to RNA-dependent RNA polymerase (RdRp) (EC: 2.7.7.48) type and performs the synthesis of both mRNAs (transcription) and genomic RNA (gRNA) (replication). In addition to the RNA polymerase, the LP also harbours two more enzymes, viz. enzymes for cap addition and cap methylation of mRNAs. The polymerase domain of the LP is analyzed for its active site amino acids and its proofreading (PR) domain. Two polymerase active site regions and a DEDD-superfamily of 3’→5’ PR exonuclease active site domain are identified in the polymerase region. The signature metal-binding motifs, viz. -GDNQ- and –SDD- which are commonly found in the RdRps of all the (-) strand RNA viral pathogens are also found in the hRSV RNA polymerase. The two highly conserved polymerase catalytic core regions identified by sequence similarity are in close agreement with other DNA/RNA polymerases already reported and hence, proposed to function in the nucleotidyl transfer reactions. Presence of the two catalytic regions also suggest that the polymerase may function in a dual mode, one for transcription and the other one for replication, using the same invariant catalytic Mg2+-binding –GDNQ- and –SDD- motifs.

Computer simulation approach to the identification of visfatin-derived angiogenic peptides.

Angiogenesis plays an essential role in various normal physiological processes, such as embryogenesis, tissue repair, and skin regeneration. Visfatin is a 52 kDa adipokine secreted by various tissues including adipocytes. It stimulates the expression of vascular endothelial growth factor (VEGF) and promotes angiogenesis. However, there are several issues in developing full-length visfatin as a therapeutic drug due to its high molecular weight. Therefore, the purpose of this study was to develop peptides, based on the active site of visfatin, with similar or superior angiogenic activity using computer simulation techniques.Initially, the active site domain (residues 181∼390) of visfatin was first truncated into small peptides using the overlapping technique. Subsequently, the 114 truncated small peptides were then subjected to molecular docking analysis using two docking programs (HADDOCK and GalaxyPepDock) to generate small peptides with the highest affinity for visfatin. Furthermore, molecular dynamics simulations (MD) were conducted to investigate the stability of the protein-ligand complexes by computing root mean square deviation (RSMD) and root mean square fluctuation(RMSF) plots for the visfatin-peptide complexes. Finally, peptides with the highest affinity were examined for angiogenic activities, such as cell migration, invasion, and tubule formation in human umbilical vein endothelial cells (HUVECs). Through the docking analysis of the 114 truncated peptides, we screened nine peptides with a high affinity for visfatin. Of these, we discovered two peptides (peptide-1: LEYKLHDFGY and peptide-2: EYKLHDFGYRGV) with the highest affinity for visfatin. In an in vitrostudy, these two peptides showed superior angiogenic activity compared to visfatin itself and stimulated mRNA expressions of visfatin and VEGF-A. These results show that the peptides generated by the protein-peptide docking simulation have a more efficient angiogenic activity than the original visfatin.

Efficiently catalytic degradation of tetracycline via persulfate activation with plant-based biochars: Insight into endogenous mineral self-template effect and pyrolysis catalysis

Endogenous mineral of plant such as potassium, calcium and iron may play a crucial role in boosting the physicochemical structure and catalytic activity of high temperature pyrolyzed plant-based biochar while it is often neglected owing to its relative less content. Herein, self-template pyrolyzed plant-based biochars were prepared from two different ash-contained agricultural wastes of peanut hull (PH, 3.2% ash) and cotton straw (CS, 0.8% ash), and aimed at investigating the relationship among the endogenous mineral fractions of plant-based biomass, physicochemical active structure and persulfate (PS) catalytic degradation activity for tetracycline (TC). The results of energy/spectral characterization showed that under the self-template effect and pyrolysis catalysis of endogenous minerals, PH biochar (PBC) possessed much more specific surface area, conjugated graphite domain, C=O and pyrrolic-N surface active functional sites than CS biochar (CBC), enhancing TC removal rate of PBC/PS to 88.37%, twice that of CBC/PS (44.16%). Meanwhile, reactive oxygen quenching and electrochemical experiments showed that electrons transfer and non-free radical pathways based on singlet oxygen contributed 92% of TC removal in PBC/PS system. Remarkably, by comparing the differences in structure and TC removal performance of pre-deashing and non-deashing prepared plant-based biochars, a possible mechanism for endogenous mineral components' self-template effect and pyrolysis catalysis role of plant-based biomass was proposed. This study provides a new insight for revealing the intrinsic mechanism of mineral elements enhancing the active surface structures and catalytic properties of plant-based biochars derived from distinct feedstocks.

P-668 Effects of the active site domain of visfatin on the improvement of ovarian function in 4-vinylcyclohexene diepoxide-induced ovarian failure mice

Abstract Study question Can only the active site of domain of visfatin improve ovarian function in mice with 4-vinylcyclohexene diepoxide (VCD)-induced ovarian failure? Summary answer The active site domain of visfatin improves ovarian function and fertility potential in VCD-induced mice by stimulating the ovarian angiogenesis and early follicular development. What is known already Ovarian aging is a representative unmet need in infertility treatment and many studies have attempted to improve oocytes quality by activating ovarian angiogenesis. Visfatin has been reported to have angiogenic activity. Our previous study reported that visfatin improved ovarian function and fertility potential in aged female mice. However, visfatin has many limitations in developing as a drug due to large molecular weight. Visfatin is composed of two structural domains, and the active site is located in the second structural domain (amino acid residues 181 to 390), which were defined as the active site domain (hereafter A-domain peptide) in this study. Study design, size, duration This is a controlled experimental study using a total of mice with 60 VCD-induced ovarian failure. The duration of this study was from January to November 2022. Participants/materials, setting, methods C57BL/6 female mice were randomly divided into six groups: Five groups received 160mg/kg VCD and one group received an equal volume of vehicle. Four VCD groups were intraperitoneally injected three times at intervals of 2 days with 500 and 1000 ng/ml of full-size visfatin and A-domain peptide, respectively. The remaining one group served as a negative control. After final treatment, follicular development, gene expression, AMH level, and fertility potential were examined. Main results and the role of chance In hematoxylin and eosin (H&E) staining, numbers of functional follicles including primordial, primary, secondary, and antral follicles were significantly decreased in the VCD-vehicle group compared to the control. However, treatment of full-size visfatin and A-domain peptide increased the number of primordial and primary follicle. On immnohistochemistry (IHC), expression of BMP15, C-KIT, VEGF, and visfatin in ovarian tissues were significantly reduced in the VCD-vehicle group, but immunoreactivity of these factors strongly stained in the treatment of full-size visfatin and A-domain peptide. This effect was more remarkable in 500 ng/ml of A-domain peptide compared to other treatments. The serum AMH level was significantly lower in the VCD-vehicle group compared to the control group, but it was increased after the treatment with A-domain peptide and full-size visfatin. Limitations, reasons for caution This study does not elucidate a clear mechanism how A-domain peptide increases the ovarian function and fertility of VCD-induced ovarian failure mice although it stimulated the expression of genes associated with early follicular development and angiogenesis. Also, generalizability to humans may be limited because the study was conducted in mice. Wider implications of the findings This study suggests that administration of A-domain peptide could be applied as a new treatment strategy for ovarian aging, presumably by activating ovarian angiogenesis and dormant primordial follicles. Trial registration number Not applicable

Biochemical, molecular and anti-tumor characterization of L-methionine gamma lyase produced by local Pseudomonas sp. in Egypt

A soil inhabiting Pseudomonas sp. has been examined for producing L- methionine gamma-lyase enzyme. The identity of the tested bacteria was verified by VITEK2, and MALDI-TOF analysis in addition to molecular confirmation by 16S rDNA sequence and submitted in Genbank under accession number ON993898.1. Production of the targeted enzyme was done using a commercial medium including L-methionine, as the main substrate. This obtained enzyme was precipitated using acetone (1:1v/v) followed by purification with Sephadex G100 and sepharose columns. The specific activity of the purified enzyme (105.8 µmol/ mg/min) increased by 1.89 folds after the purification steps. The peptide fingerprint of the native MGL was verified from the proteomics analysis, with identical conserved active site domains with database-deposited MGLs. The molecular mass of the pure MGL denatured subunit was (>40 kDa) and that of the native enzyme was (>150 kDa) ensuring their homotetrameric identity. The purified enzyme showed absorption spectra at 280 nm and 420 nm for the apo-MGL and PLP coenzyme, respectively. Amino acids suicide analogues analysis by DTNB, hydroxylamine, iodoacetate, MBTH, mercaptoethanol and guanidine thiocyanate reduced the relative activity of purified MGL. From the kinetic properties, the catalytic effectiveness (Kcat/km) of Pseudomonas sp. MGL was 10.8 mM -1 S-1 for methionine and 5.51 mM -1 S-1 for cysteine, respectively. The purified MGL showed highly significant antiproliferative activity towards the liver carcinoma cell line (HEPG-2) and breast carcinoma cell line (MCF-7) with half inhibitory concentration values (IC50) 7.23 U/ml and 21.14 U/ml, respectively. No obvious signs of toxicity on liver and kidney functions in the examined animal models were observed.

Identification and characterization of the largest deletion in the PCCA gene causing severe acute early-onset form of propionic acidemia.

Whole-exome sequencing (WES) is an excellent method for the diagnosis of diseases of uncertain or heterogeneous genetic origin. However, it has limitations for detecting structural variations such as InDels, which the bioinformatics analyzers must be aware of. This study aimed at using WES to evaluate the genetic cause of the metabolic crisis in a 3-day-old neonate admitted to the neonatal intensive care unit (NICU) and deceased after a few days. Tandem mass spectrometry (MS/MS) showed a significant increase in propionyl carnitine (C3), proposing methylmalonic acidemia (MMA) or propionic acidemia (PA). WES demonstrated a homozygous missense variant in exon 4 of the BTD gene (NM_000060.4(BTD):c.1330G > C), responsible for partial biotinidase deficiency. Segregation analysis of the BTD variant revealed the homozygous status of the asymptomatic mother. Furthermore, observation of the bam file, around genes responsible for PA or MMA, by Integrative Genomics Viewer (IGV) software displayed a homozygous large deletion in thePCCAgene. Comprehensive confirmatory studies identified and segregated a novel outframe deletion of 217,877bp length, "NG_008768.1:g.185211_403087delinsTA", extended from intron 11 to 21 of the PCCA, inducing a premature termination codon and activation of nonsense-mediated mRNA decay (NMD). Homology modeling of the mutant PCCA demonstrated eliminating the protein's active site and critical functional domains. Thereupon, this novel variant is suggested as the largest deletion in the PCCA gene, causing an acute early-onset PA. These results could expand the PCCA variants spectrum, and improve the existing knowledge on the molecular basis of PA, as well as provide new evidence of pathogenicity of the variant (NM_000060.4(BTD):c.1330G > C.

Recombinant production, purification, and biochemical characterization of a novel L-lactate dehydrogenase from Bacillus cereus NRC1 and inhibition study of mangiferin.

Lactate dehydrogenase (LDH, EC 1.1.1.27) is one of the vital glycolytic conditions, especially during anaerobic conditions. It is a significant diagnostic, prognostic, and monitoring biomarker parameter. A 950-bp DNA fragment containing the gene (LDH) encoding LDH was amplified from Bacillus cereus NRC1. The deduced amino acid sequence reveals that B. cereus LDH (Bc-LDH) is highly homologous to the LDHs of Bacillus organisms. All LDH enzymes have a significant degree of conservation in their active site and several additional domains with unidentified functions. The gene for LDH, which catalyzes lactate synthesis, was cloned, sequenced (accession number: LC706200.1), and expressed in Escherichia coli BL21 (DE3). In this investigation, Bc-LDH was purified to homogeneity with a specific activity of 22.7 units/mg protein and a molecular weight of 35kDa. It works optimally at pH 8.0. The purified enzyme was inhibited by FeCl2, CuCl2, ZnCl2, and NiCl, whereas CoCl2 was found to boost the activity of Bc-LDH. The molecular docking of the 3D model of the Bc-LDH structure with a natural inhibitor, mangiferin, demonstrated excellent LDH inhibition, with a free binding energy of -10.2kcal/mol. Moreover, mangiferin is a potent Bc-LDH inhibitor that inhibits Bc-LDH competitively and has one binding site with a Ki value of 0.075mM. The LDH-mangiferin interaction exhibits a low RMSF value (>1.5Å), indicating a stable contact at the residues. This study will pave the way for more studies to improve the understanding of mangiferin, which could be considered an intriguing candidate for creating novel and improved LDH inhibitors.

Characterization of RNA polymerase II trigger loop mutations using molecular dynamics simulations and machine learning.

Catalysis and fidelity of multisubunit RNA polymerases rely on a highly conserved active site domain called the trigger loop (TL), which achieves roles in transcription through conformational changes and interaction with NTP substrates. The mutations of TL residues cause distinct effects on catalysis including hypo- and hyperactivity and altered fidelity. We applied molecular dynamics simulation (MD) and machine learning (ML) techniques to characterize TL mutations in the Saccharomyces cerevisiae RNA Polymerase II (Pol II) system. We did so to determine relationships between individual mutations and phenotypes and to associate phenotypes with MD simulated structural alterations. Using fitness values of mutants under various stress conditions, we modeled phenotypes along a spectrum of continual values. We found that ML could predict the phenotypes with 0.68 R2 correlation from amino acid sequences alone. It was more difficult to incorporate MD data to improve predictions from machine learning, presumably because MD data is too noisy and possibly incomplete to directly infer functional phenotypes. However, a variational auto-encoder model based on the MD data allowed the clustering of mutants with different phenotypes based on structural details. Overall, we found that a subset of loss-of-function (LOF) and lethal mutations tended to increase distances of TL residues to the NTP substrate, while another subset of LOF and lethal substitutions tended to confer an increase in distances between TL and bridge helix (BH). In contrast, some of the gain-of-function (GOF) mutants appear to cause disruption of hydrophobic contacts among TL and nearby helices.

Functional and Phylogenetic Diversity of Cas10 Proteins.

Cas10 proteins are large subunits of type III CRISPR RNA (crRNA)-guided surveillance complexes, many of which have nuclease and cyclase activities. Here, we use computational and phylogenetic methods to identify and analyze 2014 Cas10 sequences from genomic and metagenomic databases. Cas10 proteins cluster into five distinct clades that mirror previously established CRISPR-Cas subtypes. Most Cas10 proteins (85.0%) have conserved polymerase active-site motifs, while HD-nuclease domains are less well conserved (36.0%). We identify Cas10 variants that are split over multiple genes or genetically fused to nucleases activated by cyclic nucleotides (i.e., NucC) or components of toxin-antitoxin systems (i.e., AbiEii). To clarify the functional diversification of Cas10 proteins, we cloned, expressed, and purified five representatives from three phylogenetically distinct clades. None of the Cas10s are functional cyclases in isolation, and activity assays performed with polymerase domain active site mutants indicate that previously reported Cas10 DNA-polymerase activity may be a result of contamination. Collectively, this work helps clarify the phylogenetic and functional diversity of Cas10 proteins in type III CRISPR systems.

Analysis of RNA polymerase II trigger loop mutations using simulations and machine learning.

RNA polymerase catalyzes RNA synthesis by the help of the coordinated actions of a near active site domain called the trigger loop (TL). TL is documented to have functions in nucleotide selection, positioning, fidelity and backtracking, while the mechanisms of these events and the exact roles of TL are unknown. In this study, we performed a comprehensive analysis of the TL mutations using molecular dynamics (MD) simulations and deep learning techniques. We generated a numerical continuum of the complete phenotypical map of the TL mutants using a second generation of genetic fitness data by a deep learning approach. We showed that amino acid sequences of the TL mutants could predict the continuous phenotypes at 0.68 R2 correlation. Incorporation of the structural data from the MD simulations into these predictive models has a minor effect in the predictions. On the other hand, MD data brought important insights on the structural outcomes of the distinct phenotypes that are clustered based on a variational autoencoder (VAE) model. This study showed that most of the lethal and some of the loss-of-function (LOF) mutants have large distances between the near active site residues and the incoming nucleoside triphosphate (NTP) suggesting a direct mechanism for affecting the catalysis, while a subset of LOF mutants manifested closer distances for NTP that suggests a more indirect mechanism. We showed that mutants with the gain-of-function (GOF) phenotype directly or indirectly interrupted a hydrophobic pocket previously known as stabilizing the open TL. Overall, this study provides a systematic description of structural outcomes of the distinct TL mutations.

Infancy‐onset diabetes caused by de‐regulated AMPylation of the human endoplasmic reticulum chaperone BiP

Dysfunction of the endoplasmic reticulum (ER) in insulin‐producing beta cells results in cell loss and diabetes mellitus. Here we report on five individuals from three different consanguineous families with infancy‐onset diabetes mellitus and severe neurodevelopmental delay caused by a homozygous p.(Arg371Ser) mutation in FICD. The FICD gene encodes a bifunctional Fic domain‐containing enzyme that regulates the ER Hsp70 chaperone, BiP, via catalysis of two antagonistic reactions: inhibitory AMPylation and stimulatory deAMPylation of BiP. Arg371 is a conserved residue in the Fic domain active site. The FICDR371S mutation partially compromises BiP AMPylation in vitro but eliminates all detectable deAMPylation activity. Overexpression of FICDR371S or knock‐in of the mutation at the FICD locus of stressed CHO cells results in inappropriately elevated levels of AMPylated BiP and compromised secretion. These findings, guided by human genetics, highlight the destructive consequences of de‐regulated BiP AMPylation and raise the prospect of tuning FICD's antagonistic activities towards therapeutic ends.