Catalytic Function Research Articles

Occurrence of protease-like catalytic activity in the polyclonal IgG in schizophrenia and bipolar disorder.

Neuro-immune dysfunction and inflammation are said to be involved in the aetiology of major mental illnesses. This study describes the assessment of the protease-like catalytic function of serum IgG in psychiatric disorders, schizophrenia and bipolar disorder, along with healthy controls of Indian ethnicity. Systemic lupus erythematosus patients experiencing neuropsychiatry conditions were included as comparators for the comprehensive evaluation of IgG catalytic function. The catalytic activity of serum IgG was determined using the generic peptide substrate Pro-Phe-Arg-methylcoumarylamide, and then the apparent kinetic data was computed. Compared to healthy controls (n = 25), the activity of those with schizophrenia (n = 30) was found to be 11-fold higher. The neuropsychiatry lupus patients (n = 25) exhibited 2.7 times less activity than the schizophrenia group. The IgG activity of the bipolar patients' (n = 30) were found to be 2.9 and 12 times higher than lupus and healthy controls. IgG activity showed a modest, no significant, link with PANSS positive and negative disease scores, in a subset of schizophrenia patients. The study's findings express presence of catalytic antibodies in the blood as well as neuro-immune dysfunction in major psychosis disorders. In addition, subsets of schizophrenia patients indicate presence of autoimmune component in them.

Catalytic Enantioselective Functionalization of Maleimides: An Update

Comprehensive SummaryMaleimide derivatives are well‐established reactive intermediates also found in natural products, synthetic pharmaceuticals and functional polymers. Their specific reactivity found widespread applications in the field of bioconjugation and allowed for the development of highly selective functionalizations based on simple additions and cycloadditions with the possible control of central and C–N axial chirality. These multisite‐reactive scaffolds have aroused a long‐standing interest throughout the scientific community more particularly as powerful electrophilic partners and more recently as nucleophilic partners in some specific transformations. The persistent interest in these easily accessible synthetic platforms over the last decade has enabled the development of new enantioselective transformations and the major advancements in this field are presented in this review. Key Scientists

Computational Stabilization of a Non‐Heme Iron Enzyme Enables Efficient Evolution of New Function

AbstractDeep learning tools for enzyme design are rapidly emerging, and there is a critical need to evaluate their effectiveness in engineering workflows. Here we show that the deep learning‐based tool ProteinMPNN can be used to redesign Fe(II)/αKG superfamily enzymes for greater stability, solubility, and expression while retaining both native activity and industrially relevant non‐native functions. This superfamily has diverse catalytic functions and could provide a rich new source of biocatalysts for synthesis and industrial processes. Through systematic comparisons of directed evolution trajectories for a non‐native, remote C(sp3)−H hydroxylation reaction, we demonstrate that the stabilized redesign can be evolved more efficiently than the wild‐type enzyme. After three rounds of directed evolution, we obtained a 6‐fold activity increase from the wild‐type parent and an 80‐fold increase from the stabilized variant. To generate the initial stabilized variant, we identified multiple structural and sequence constraints to preserve catalytic function. We applied these criteria to produce stabilized, catalytically active variants of a second Fe(II)/αKG enzyme, suggesting that the approach is generalizable to additional members of the Fe(II)/αKG superfamily. ProteinMPNN is user‐friendly and widely accessible, and our results provide a framework for the routine implementation of deep learning‐based protein stabilization tools in directed evolution workflows for novel biocatalysts.

Nuclear localization of MTHFD2 is required for correct mitosis progression.

Subcellular compartmentalization of metabolic enzymes establishes a unique metabolic environment that elicits specific cellular functions. Indeed, the nuclear translocation of certain metabolic enzymes is required for epigenetic regulation and gene expression control. Here, we show that the nuclear localization of the mitochondrial enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) ensures mitosis progression. Nuclear MTHFD2 interacts with proteins involved in mitosis regulation and centromere stability, including the methyltransferases KMT5A and DNMT3B. Loss of MTHFD2 induces severe methylation defects and impedes correct mitosis completion. MTHFD2 deficient cells display chromosome congression and segregation defects and accumulate chromosomal aberrations. Blocking the catalytic nuclear function of MTHFD2 recapitulates the phenotype observed in MTHFD2 deficient cells, whereas restricting MTHFD2 to the nucleus is sufficient to ensure correct mitotic progression. Our discovery uncovers a nuclear role for MTHFD2, supporting the notion that translocation of metabolic enzymes to the nucleus is required to meet precise chromatin needs.

Aesthetic Literacies in Digital Learning Ecologies: Tracing Aesthetic Tools and Functions in a Youth Literary Salon

Concerned with disinformation, fake news, and a posttruth era, literacy research on digital learning ecologies has focused on content of the texts that youth encounter but less explicitly on aesthetics—issues of form or the way a text is written. Drawing on critical sociocultural theories, this article examines youth aesthetic meaning-making with non-neutral digital texts. The data analyzed are part of a multiyear multimethod study of the aesthetic literacy practices of queer youth of color and allies. Data were collected during an online summer literary salon where youth discussed textual content alongside issues of aesthetic forms. Findings illustrate that youth drew on a broad range of aesthetic tools to achieve (1) a poetic function concerned with how aesthetic forms elicit figurative, connotative, and storied meanings; (2) a catalytic function concerned with how aesthetic forms persuade, manipulate, or encourage particular actions; and (3) an ideological function concerned with how aesthetic forms inscribe or contest social inequalities. These tools and functions, complicate previous understandings of disinformation, fake news, and critical reasoning in literacies research. This study suggests a critical sociocultural approach to aesthetics as territory for further scholarship across many areas of literacy research.

UBR7 E3 Ligase Suppresses Interferon-β Mediated Immune Signaling by Targeting Sp110 in Hepatitis B Virus-Induced Hepatocellular Carcinoma.

A newly discovered E3 ubiquitin ligase, UBR7, plays a crucial role in histone H2BK120 monoubiquitination. Here, we report a novel function of UBR7 in promoting hepatitis B virus (HBV) pathogenesis, which further leads to HBV-induced hepatocellular carcinoma (HCC). Transcriptomics analysis from HCC patients revealed the deregulation of UBR7 in cancer. Remarkably, targeting UBR7, particularly its catalytic function, led to a significant decrease in viral copy numbers. We also identified the speckled family protein Sp110 as an important substrate of UBR7. Notably, Sp110 has been previously shown to be a resident of promyelocytic leukemia nuclear bodies (PML-NBs), where it remains SUMOylated, and during HBV infection, it undergoes deSUMOylation and exits the PML body. We observed that UBR7 ubiquitinates Sp110 at critical residues within its SAND domain. Sp110 ubiquitination downregulates genes in the type I interferon response pathway. Comparative analysis of RNA-Seq from the UBR7/Sp110 knockdown data set confirmed that the IFN-β signaling pathway gets deregulated in HCC cells in the presence of HBV. Single-cell RNA-Seq analysis of patient samples further confirmed the inverse correlation between the expression of Sp110/UBR7 and the inflammation score. Notably, silencing of UBR7 induces IRF7 phosphorylation, thereby augmenting interferon (IFN)-β and the downstream interferon-stimulated genes (ISGs). Further, wild-type but not the ubiquitination-defective mutant of Sp110 could be recruited to the type I interferon response pathway genes. Our study establishes a new function of UBR7 in non-histone protein ubiquitination, promoting viral persistence, and has important implications for the development of therapeutic strategies targeting HBV-induced HCC.

Repurposing Tolfenamic Acid to Anchor the Uncharacterized Pocket of the PUB Domain for Proteolysis of the Atypical E3 Ligase HOIP.

The E3 ligase HOIP is vital for the NF-κB pathway and is implicated in cancer and immunity. However, it remains challenging to achieve high selectivity by directly targeting the conserved catalytic RBR domain of HOIP. Herein, we identified four low-molecular-weight compounds that bind to an uncharacterized pocket of the HOIP PUB domain (HOIPPUB). The complex structure facilitated the discovery of the first single-digit micromolar ligand of HOIPPUB, tolfenamic acid, which exhibited over 30-fold selectivity due to the low sequence identity of the uncharacterized pocket of HOIPPUB. Although tolfenamic acid did not block the substrate recognition and linear ubiquitination activity of HOIP, a ligand of the uncharacterized PUB pocket of HOIP (LUPH), by chemical linking pomalidomide with tolfenamic acid, degraded HOIP, reduced NEMO ubiquitination and p65 phosphorylation, and eventually inhibited NF-κB activation and breast cancer cell proliferation. Our work proposes an alternative strategy to target the nonfunctional pocket of the PUB domain with high sequence diversity to promote HOIP degradation, rather than targeting the conserved RBR domain to block the catalytic function of HOIP.

Targeting N-methyl-lysine histone demethylase KDM4 in cancer: natural products inhibitors as a driving force for epigenetic drug discovery.

KDM4A-F enzymes are a subfamily of histone demethylases containing the Jumonji C domain (JmjC) using Fe(II) and 2-oxoglutarate for their catalytic function. KDM4 enzymes are overexpressed or deregulated in cancer, thus leading to alteration in chromatin structure and transcriptional defects. As KDM4 enzymes have been associated with malignancy, they may represent novel targets for developing innovative therapeutic tools to treat different solid and blood tumors, of note, KDM4A is the isozyme most frequently associated with aggressive phenotypes of these tumors. To this aim, industrial and academic medicinal chemistry efforts have identified different KDM4 inhibitors. In most cases, these are pan-KDM4 inhibitors, with low selectivity against other Jumonji family members. The pharmacophoric features of the inhibitors frequently include a chelating group capable of coordinating the catalytic iron within the active site of the KDM4 enzyme. Nonetheless, non-chelating compounds have also demonstrated promising inhibitory activity, suggesting potential flexibility in the drug design. Several natural products, containing monovalent or bivalent chelators, have been identified as KDM4 inhibitors, albeit with a micromolar inhibition potency. This highlights the potential for leveraging them as templates for the design and synthesis of new derivatives, exploiting nature's chemical diversity to pursue more potent and selective KDM4 inhibitors.

Mutations Selectively Evolving Peroxidase Activity Among Alternative Catalytic Functions of Human Glutathione Transferase P1-1

Glutathione transferases are detoxication enzymes with broad catalytic diversity, and small alterations to the protein’s primary structure can have considerable effects on the enzyme’s substrate selectivity profile. We demonstrate that two point mutations in glutathione transferase P1-1 suffice to generate 20-fold enhanced non-selenium-dependent peroxidase activity indicating a facile evolutionary trajectory. Designed mutant libraries of the enzyme were screened for catalytic activities with alternative substrates representing four divergent chemistries. The chemical reactions comprised aromatic substitution, Michael addition, thiocarbamoylation, and hydroperoxide reduction. Two mutants, R1 (Y109H) and an R1-based mutant V2 (Q40M-E41Q-A46S-Y109H-V200L), were discovered with 16.3- and 30-foldincreased peroxidase activity with cumene hydroperoxide (CuOOH) compared to the wildtype enzyme, respectively. The basis of the improved peroxidase activity of the mutant V2 was elucidated by constructing double-point mutants. The mutants V501 (Q40M-Y109H) and V503 (E41Q-Y109H) were found to have 20- and 21-fold improvements in peroxidase activity relative to the wildtype enzyme, respectively. The steady-state kinetic profiles of mutants R1 and V2 in the reduction of CuOOH were compared to the wildtype parameters. The kcat values for R1 and V2 were 34- and 57-fold higher, respectively, than that of the wildtype enzyme, whereas the mutant Km values were increased approximately 3-fold. A 10-fold increased catalytic efficiency (kcat/Km) in CuOOH reduction is accomplished by the Tyr109His point mutation in R1. The 23-fold increase of the efficiency obtained in V2 was caused by adding further mutations primarily enhancing kcat. In all mutants with elevated peroxidase activity, His109 played a pivotal role.

Cobalt-Zirconia coated monoliths fabricated by Direct Ink Writing for catalytic applications

Direct Ink Writing (DIW) is a promising technique for fabricating ceramic catalysts with complex geometries and enhanced catalytic performances, but these still require overcoming some challenges to maximize their catalytic and mechanical functionality. In this work, DIW yttria-doped zirconia (YDZ) catalysts were coated with a Co/YDZ catalyst layer. Two configurations based on a dense monolith and a porous one sintered at 1450ºC and 1300ºC were fabricated through DIW, using optimal rheological hydrogel-inks, to investigate the effect of porosity on the mechanical and catalytic performance in the ethanol steam reforming reaction. The monolith sintered at 1450°C achieved the best mechanical and catalytic performance, as it presented the highest Co concentration (2.3at.%) at the catalyst layer. In addition, this monolith exhibited excellent mechanical properties at the interface between contiguous filaments in the multilayer structure. Therefore, this simple approach is a promising method to produce enhanced catalytically and mechanically robust monoliths.

Purification and catalysis of choline dehydrogenase from Escherichia coli

Choline dehydrogenase (CHDH) is a membrane-bound enzyme belonging to the glucose-methanol-choline (GMC) oxidoreductase superfamily, which is characterized by a crucial FAD-binding domain essential for catalytic function. CHDH catalyzes the oxidation of choline to betaine aldehyde, which is further oxidized to betaine, a vital osmoprotectant and methyl donor for cellular physiology and metabolism. However, the detailed catalytic mechanism of CHDH still remains poorly understood. In our investigation, we gained purity E. coli CHDH samples in DDM (n-dodecyl-β-D-maltoside) and SMA (styrene maleic acid) copolymer respectively and examined their structural composition and catalytic activity separately. Our findings demonstrated the effectiveness of SMA, commonly employed for extracting transmembrane proteins and can preserve the natural bio-membrane environment surrounding the enzyme, in extracting peripheral membrane proteins like CHDH here, which lacks transmembrane helices. CHDH exhibited a trimeric conformation in SMA, whereas it existed as monomers in DDM, as determined by our negative staining analysis. Our experiments also revealed that highly pure E. coli CHDH could only oxidize choline to betaine aldehyde but failed to further oxidize betaine aldehyde to betaine as determined by the biochemical and enzymatic reaction kinetic assays. In addition, the enzyme in SMA displayed greater catalytic activity compared to that in DDM. Furthermore, we confirmed the crucial role of His473, which is hypothesized to be a critical site for substrate binding from our structural comparative analysis between CHDH and its highly homologous choline oxidase, in the catalytic activity of the enzyme through gene mutation. Our work also sheds light on CHDH’s contribution to cellular osmotic tolerance through gene knockout. This research enhances our better understanding of CHDH within cellular biochemistry and metabolic pathways.

Evolutionary Analysis and Catalytic Function of LOG Proteins in Plants

Background: The plant hormone cytokinin is a conserved regulator of plant development. LONELY GUY (LOG) proteins are pivotal in cytokinin biosynthesis. However, their origin, evolutionary history, and enzymatic characteristics remain largely uncharacterized. Methods: To elucidate LOG family evolution history and protein motif composition, we conducted phylogenetic and motif analyses encompassing representative species across the whole green plant lineage. Catalytic activity and structure analysis were conducted to thoroughly characterize the LOG proteins. Results: Our phylogeny showed that LOG proteins could be divided into five groups and revealed three major duplication events giving rise to four distinct groups of vascular LOG proteins. LOG proteins share a conserved structure characterized by a canonical motif arrangement comprising motifs 1, 2, 3, 4, 5, 6, and 7. Two significant changes in LOG motif composition occurred during the transition to land plants: the emergence of motif 3 in charophyte LOG sequences and the subsequent acquisition of motif 8 at the C-terminus of LOG proteins. Enzymatic assays demonstrated that LOG proteins can be classified into two groups based on their enzyme activity. One group act as cytokinin riboside 5′-monophosphate phosphoribohydrolase and primarily convert iPRMP to iP, while the other group act as 5′-ribonucleotide phosphohydrolase, and preferentially produce iPR from the same substrates. TaLOG5-4A1, TaLOG5-4A2, TaLOG5-5B2, and TaLOG5-D1 shared conserved residues in the critical motif and were predicted to have similar protein structures, but displayed distinct enzymatic activities. Conclusions: Our findings provide a comprehensive overview of LOG protein phylogeny and lay a foundation for further investigations into their functional diversification.

Recognition-Encoded Molecules: A Minimal Self-Replicator.

Nucleic acids, with their unique duplex structure, which is key for information replication, have sparked interest in self-replication's role in life's origins. Early template-based replicators, initially built on short oligonucleotides, expanded to include peptides and synthetic molecules. We explore here the potential of a class of synthetic duplex-forming oligoanilines, as self-replicators. We have recently developed oligoanilines equipped with 2-trifluoromethylphenol-phosphine oxide H-bond base pairs and we investigate whether the imine formed between aniline and aldehyde complementary monomers can self-replicate. Despite lacking a clear sigmoidal kinetic profile, control experiments with a methylated donor and a competitive inhibitor support self-replication. Further investigations with the reduced aniline dimer demonstrate templated synthesis, revealing a characteristic parabolic growth. After showing sequence selective duplex formation, templated synthesis and the emergence of catalytic function, the self-replication behaviour further suggests that the unique properties of nucleic acids can be paralleled by synthetic recognition-encoded molecules.

NTF2‐Like Enzymes as Versatile Biocatalysts in Fungal Natural Product Biosynthesis

AbstractFungal natural products (NPs), known for their potent bioactivities, can be utilized as human therapeutics and agrochemicals. These bioactive and structurally complex compounds are biosynthesized through condensation of monomeric building blocks to construct core scaffolds, followed by various modification steps. Recent studies have revealed that a unique class of enzymes from the NTF2‐like protein family plays important roles in the biosynthesis of complex fungal NPs. These NTF2‐like enzymes belong to a large group of related proteins that share a common fold with nuclear transport factor 2, and are capable of catalyzing various reactions. In this study, we summarize the recent progress in discovering and characterizing the catalytic functions of fungal‐derived NTF2‐like enzymes, including dehydratases, epimerases, isomerases, semipinacolases, pericyclases, and aldolases. These findings not only provide valuable insights into new catalytic reactions and mechanisms, but also offer opportunities to discover novel NPs and biocatalysts through genome mining.

Unveiling the Activity Origin of Electrochemical Oxygen Evolution on Heteroatom‐Decorated Carbon Matrix

AbstractChemical modification via functional dopants in carbon materials holds great promise for elevating catalytic activity and stability. To gain comprehensive insights into the pivotal mechanisms and establish structure‐performance relationships, especially concerning the roles of dopants, remains a pressing need. Herein, we employ computational simulations to unravel the catalytic function of heteroatoms in the acidic oxygen evolution reaction (OER), focusing on a physical model of high‐electronegative F and N co‐doped carbon matrix. Theoretical and experimental findings elucidate that the enhanced activity originates from the F and pyridinic‐N (Py−N) species that achieve carbon activation. This activated carbon significantly lowers the conversion energy barrier from O* to OOH*, shifts the potential‐limiting step from OOH* formation to O* generation, and ultimately optimizes the energy barrier of the potential‐limiting step. This wok elucidates that the critical role of heteroatoms in catalyzing the reaction and unlocks the potential of carbon materials for acidic OER.

Inversions encounter relaxed genetic constraints and balance birth and death of TPS genes in Curcuma

Evolutionary dynamics of inversion and its impact on biochemical traits are a puzzling question. Here, we show abundance of inversions in three Curcuma species (turmeric, hidden ginger and Siam tulip). Genes within inversions display higher long terminal repeat content and lower expression level compared with genomic background, suggesting inversions in Curcuma experience relaxed genetic constraints. It is corroborated by depletion of selected SNPs and enrichment of deleterious mutations in inversions detected among 56 Siam tulip cultivars. Functional verification of tandem duplicated terpene synthase (TPS) genes reveals that genes within inversions become pseudogenes, while genes outside retain catalytic function. Our findings suggest that inversions act as a counteracting force against tandem duplication in balancing birth and death of TPS genes and modulating terpenoid contents in Curcuma. This study provides an empirical example that inversions are likely not adaptive but affect biochemical traits.

Unveiling the Activity Origin of Electrochemical Oxygen Evolution on Heteroatom-Decorated Carbon Matrix.

Chemical modification via functional dopants in carbon materials holds great promise for elevating catalytic activity and stability. To gain comprehensive insights into the pivotal mechanisms and establish structure-performance relationships, especially concerning the roles of dopants, remains a pressing need. Herein, we employ computational simulations to unravel the catalytic function of heteroatoms in the acidic oxygen evolution reaction (OER), focusing on a physical model of high-electronegative F and N co-doped carbon matrix. Theoretical and experimental findings elucidate that the enhanced activity originates from the F and pyridinic-N (Py-N) species that achieve carbon activation. This activated carbon significantly lowers the conversion energy barrier from O* to OOH*, shifts the potential-limiting step from OOH* formation to O* generation, and ultimately optimizes the energy barrier of the potential-limiting step. This wok elucidates that the critical role of heteroatoms in catalyzing the reaction and unlocks the potential of carbon materials for acidic OER.

Catalytic function of the laccase enzyme in response to chlorpyrifos and 2,4-dichlorophenoxyacetic acid: behavior in controlled and simulated environments.

Enzymes secreted by white-rot fungi, such as laccase, offer a promising solution for treating xenobiotic compounds dangerous to the environment and human health. This study aimed to perform a comprehensive analysis of the tolerance of Pleurotus pulmonarius LBM 105 and its laccase activity toward the pesticides 2,4-D and chlorpyrifos both in vitro and in silico. The fungal strain was able to grow in different concentrations of the pesticides, showing evident morphological alterations. Laccase activity and a 53kDa electromorph were present in all treatments, showing significant stability with peak activity achieved at a pH of 5.6 and within a temperature range of 50-60°C. Three laccase genes were mapped, annotated, and characterized from the genome. PplacI obtained better structural validation and affinity energy of - 5.05 and - 7.65kcalmol-1 with 2,4-D and chlorpyrifos, respectively. The Molecular Mechanics/Poisson-Boltzmann Surface Area analysis at 250ns confirmed the docking results, revealing the existence of stronger hydrophobic interactions between laccase and chlorpyrifos and highlighting the importance of the Phe341 residue in stabilizing both complexes. Understanding the impact of pesticides on laccase's catalytic function is key to formulating and applying future biotechnological strategies with this enzyme.

Imidazole Headgroup Phospholipid Shows Asymmetric Distribution in Vesicles and Zinc-Dependent Esterase Activity

Artificial lipids have become increasingly important in generating novel nanoenzymes and nanoparticles. Imidazole has been well established as a versatile catalyst in synthetic chemistry and through its related amino acid histidine in enzymes. By exploiting the transphosphatidylation reaction of phospholipase D, the choline headgroup of phosphatidyl choline was exchanged for the imidazole moiety containing histidinol. Here, we introduce a novel phosphatidylhistidinol (PtdHisOH) lipid and characterise it with respect to its catalytic abilities and its ability to modulate vesicle size. Our data reveal a zinc-dependent esterase activity that was strongest in vesicles and micelles, with slower catalytic rates being observed in flat lipid presentation systems and two-phase systems, indicating the importance of surface presentation and curvature effects on the catalytic activity of PtdHisOH. Such lipids offer the opportunity to impart de novo catalytic functionality to self-assembled lipid systems such as synthetic cells, leading to the development of new technologies for biocatalysis applications.

Identification and Characterization of a Fungal Monoterpene Synthase Responsible for the Biosynthesis of Geraniol.

Geraniol, an acyclic monoterpenoid of substantial value extracted from the essential oils of various aromatic plants, holds significant commercial and industrial importance in the realms of food, cosmetics, medicine, and bioenergy. Geraniol synthase, which is responsible for geraniol production, has been identified in only several plant species to date. Here, we present the first cloning and characterization of a geraniol synthase (PgfTPS) from Penicillium griseofulvum. This enzyme demonstrates pronounced specificity in catalyzing the conversion of geranyl diphosphate into geraniol. Moreover, through protein modeling and site-directed mutagenesis, we have identified key active-site residues crucial for the catalytic function of PgfTPS. Finally, we utilized engineered Saccharomyces cerevisiae as a host for PgfTPS expression to facilitate geraniol production. Our findings not only advance the development of efficient biocatalysts for geraniol generation but also establish a fundamental basis for further exploration into fungal monoterpene biosynthesis.