Catalytic Specificity Research Articles

Examining prestructured β-actin peptides as substrates of histidine methyltransferase SETD3

The Nτ-His73 methylation of β-actin by histidine methyltransferase SETD3 is required for the integrity of the cellular cytoskeleton. Modulation of SETD3 activity in human cells facilitates cancer-like changes to the cell phenotype. SETD3 binds β-actin in an extended conformation, with a conserved bend-like motif surrounding His73. Here, we report on the catalytic specificity of SETD3 towards i, i + 3 stapled β-actin peptides possessing a limited conformational freedom surrounding the His73 substrate residue via positions Glu72 and Ile75. Stapled β-actin peptides were observed to be methylated less efficiently than the linear β-actin peptide. None of the stapled β-actin peptides efficiently inhibited the SETD3-catalyzed Nτ-His73 methylation reaction. Molecular dynamics simulations demonstrated that the unbound and SETD3-bound β-actin peptides display different backbone flexibility and bend-like conformations, highlighting their important role in substrate binding and catalysis. Overall, these findings suggest that reduced backbone flexibility of β-actin prevents the formation of optimal protein-peptide interactions between the enzyme and substrate, highlighting that the backbone flexibility needs to be considered when designing β-actin-based probes and inhibitors of biomedically important SETD3.

A Novel A105Y Mutant of CYP17A1 Exhibits Almost Perfect Regioselectivity in the Production of 17α-Hydroxyprogesterone.

17α-Hydroxyprogesterone (17α-OHP) is a steroid hormone with significant biological activity that can be obtained by catalyzing progesterone (PROG), the main product of sitosterol, through CYP17A1. However, increasing the catalytic specificity of HCYP17A1 for C17 hydroxylation of progesterone (PROG) poses a formidable challenge due to the close proximity of the C16 and C17 positions. In this study, a rational design was utilized to alter the spatial configuration of the substrate channel, leading to the complete abolition of its 16-hydroxylation activity. Subsequent molecular dynamics simulations revealed that the A105Y mutation heightened the rigidity of the G95-I112 region of CYP17A1, consequently regulating the direction of the entry of PROG into the catalytic pocket. Moreover, the establishment of hydrogen bonding between Y105 and R239, coupled with Pi-stacking of A105Y with F114, effectively immobilizes the substrate PROG in a fixed position, explaining the practically perfect regioselectivity observed in A105Y. Finally, a multifaceted enzymatic cascade system, incorporating A105Y, cytochrome P450 reductase (CPR), and glucose-6-phosphate dehydrogenase (ZWF) for NADPH cofactor regeneration, was constructed in Pichia pastoris GS115. The resulting biocatalyst produced 106 ± 3.2 mg L-1 17α-OHP, a 4.6-fold increase compared with A105Y alone. Thus, this study provides valuable insights for improving the regioselectivity and activity of P450 enzymes.

Catalytic activities of wild-type C. elegans DAF-2 kinase and dauer-associated mutants.

DAF-2, the Caenorhabditis elegans insulin-like receptor homolog, regulates larval development, metabolism, stress response, and lifespan. The availability of numerous daf-2 mutant alleles has made it possible to elucidate the genetic mechanisms underlying these physiological processes. The DAF-2 pathway is significantly conserved with the human insulin/IGF-1 signaling pathway; it includes proteins homologous to human IRS, GRB-2, and PI3K, making it an important model to investigate human pathological conditions. We expressed and purified the kinase domain of wild-type DAF-2 to examine the catalytic activity and substrate specificity of the enzyme. Like the human insulin receptor kinase, DAF-2 kinase phosphorylates tyrosines within specific YxN or YxxM motifs, which are important for recruiting downstream effectors. DAF-2 kinase phosphorylated peptides derived from the YxxM and YxN motifs located in the C-terminal extension of the receptor tyrosine kinase, consistent with the idea that the DAF-2 receptor may possess independent signaling capacity. Unlike the human insulin or IGF-1 receptor kinases, DAF-2 kinase was poorly inhibited by the small-molecule inhibitor linsitinib. We also expressed and purified mutant kinases corresponding to daf-2 alleles that result in partial loss-of-function phenotypes in C. elegans. These mutations caused a complete loss of kinase function in vitro. Our biochemical investigations provide new insights into DAF-2 kinase function, and the approach should be useful for studying other mutations to shed light on DAF-2 signaling in C. elegans physiology.

Sustainable di-functional stimuli-responsive imprinted nanozymes for reversible colorimetric sensing of tetracycline

Regenerable nanozymes with high catalytic specificity and sustainability may serve as substitutes for naturally occurring enzymes, but their application is hindered by inadequate and nonselective catalytic activity. In this study, we constructed a photo-responsive molecularly imprinted layer on the Fe3O4 nanozyme surface (P-MIPs) using an ATRP method and obtained high-specificity reversible colorimetric sensing of tetracycline (TC). Due to the photo-responsive properties of P-MIPs, the imprinted nanozyme undergoes reversible release and uptake of TC under alternating irradiation at 365/440 nm based on the photo-regulated conformational transformation of the 4-[(4-methacryloyloxy)phenylazo] benzenesulfonic acid (MAPASA) at the polymer receptor sites, which may be confirmed by calculating the electron densities of the trans-/cis-MAPASA forms. Simultaneously, these processes can be visualized through the change in the color of the substrates. Highly specific P-MIPs-based nanozyme also exhibit adequate stability and sustainable catalytic activity after multiple cycles. This method can be used to achieve nanozymes with high selectivity for the enrichment, separation, and detection of hazardous substances and contribute to material sustainability.

Discovery of a Glyphosate Oxidase in Nature.

Glyphosate is the most used herbicide on Earth. After a half-century of use we know only two biodegradative pathways, each of which appears to degrade glyphosate incidentally. One pathway begins with oxidation of glyphosate catalyzed by glycine oxidase (GO). To date, no naturally-occurring GO enzymes preferentially oxidize glyphosate but nonetheless are sufficiently active to initiate its degradation. However, GO enzymes that preferentially oxidize glyphosate over glycine - i.e. glyphosate oxidases - may have evolved in environments facing prolonged glyphosate exposure. To test this hypothesis, we screened a metagenomic library from glyphosate-exposed agricultural soil and identified a glyphosate oxidase from clone 11AW19 (GO19) that prefers glyphosate over glycine by four orders of magnitude. This is the first GO isolated from a natural source exhibiting a glyphosate preference. Not only have we discovered the first glyphosate oxidase in nature, but we have also demonstrated the utility of functional metagenomics to find a glyphosate oxidase with greater catalytic efficiency and specificity than those engineered using directed evolution.

Challenges and perspectives in using unspecific peroxygenases for organic synthesis

In the past 20 years, unspecific peroxygenases (UPOs) have emerged as promising biocatalysts for various organic transformations. Particularly, we have witnessed great attention being paid to the screening of new enzymes and expansion of the substrates/products. However, challenges such as enzyme stability, low turnover numbers, and substrate specificity hinder their widespread utilization in practical organic synthesis. This review article provides a concrete and mini-overview of the challenges associated with using UPOs in organic synthesis and discusses strategies for enzyme engineering to overcome these limitations. The article highlights recent advancements in UPO research and presents potential solutions to enhance their catalytic efficiency, stability, substrate specificity, and regioselectivity. Additionally, the review outlines the current methodologies employed for directed evolution and protein engineering of UPOs, along with computational modeling approaches for rational enzyme design. By addressing the challenges and exploring avenues for enzyme engineering, this review aims to shed light on the prospects of UPOs in organic synthesis.

Engineering Bacterial Chitinases for Industrial Application: From Protein Engineering to Bacterial Strains Mutation! A Comprehensive Review of Physical, Molecular, and Computational Approaches.

Bacterial chitinases are integral in breaking down chitin, the natural polymer in crustacean and insect exoskeletons. Their increasing utilization across various sectors such as agriculture, waste management, biotechnology, food processing, and pharmaceutical industries highlights their significance as biocatalysts. The current review investigates various scientific strategies to maximize the efficiency and production of bacterial chitinases for industrial use. Our goal is to optimize the heterologous production process using physical, molecular, and computational tools. Physical methods focus on isolating, purifying, and characterizing chitinases from various sources to ensure optimal conditions for maximum enzyme activity. Molecular techniques involve gene cloning, site-directed mutation, and CRISPR-Cas9 gene editing as an approach for creating chitinases with improved catalytic activity, substrate specificity, and stability. Computational approaches use molecular modeling, docking, and simulation techniques to accurately predict enzyme-substrate interactions and enhance chitinase variants' design. Integrating multidisciplinary strategies enables the development of highly efficient chitinases tailored for specific industrial applications. This review summarizes current knowledge and advances in chitinase engineering to serve as an indispensable guideline for researchers and industrialists seeking to optimize chitinase production for various uses.

Role of paraoxonase 1 in organophosphate G-series nerve agent poisoning and future therapeutic strategies.

Chemical warfare nerve agents (CWNA) are neurotoxic chemicals unethically used as agents of mass destruction by terrorist outfits and during war. The available antidote against CWNA-mediated toxicity is not sufficiently effective and possesses several limitations. As a countermeasure, paraoxonase 1 (PON1), a catalytic bioscavenger, is being developed as a prophylactic treatment. However, the catalytic activity and substrate specificity of human PON1 are insufficient to be used as a potential antidote. Several laboratories have made different approaches to enhance the CWNA hydrolytic activity against various nerve agents. This review explores the holistic view of PON1 as a potential prophylactic agent against G-series CWNA poisoning, from its initial development to recent advancements and limitations. Apart from this, the review also provides an overview of all available PON1 variants that could be used as a potential prophylactic agent and discusses several possible ways to counteract immunogenicity.

The crystal structure of Grindelia robusta 7,13-copalyl diphosphate synthase reveals active site features controlling catalytic specificity

Diterpenoid natural products serve critical functions in plant development and ecological adaptation and many diterpenoids have economic value as bioproducts. The family of class II diterpene synthases catalyzes the committed reactions in diterpenoid biosynthesis, converting a common geranylgeranyl diphosphate precursor into different bicyclic prenyl diphosphate scaffolds. Enzymatic rearrangement and modification of these precursors generates the diversity of bioactive diterpenoids. We report the crystal structure of Grindelia robusta 7,13-copalyl diphosphate synthase, GrTPS2, at 2.1 Å of resolution. GrTPS2 catalyzes the committed reaction in the biosynthesis of grindelic acid, which represents the signature metabolite in species of gumweed (Grindelia spp., Asteraceae). Grindelic acid has been explored as a potential source for drug leads and biofuel production. The GrTPS2 crystal structure adopts the conserved three-domain fold of class II diterpene synthases featuring a functional active site in the γβ-domain and a vestigial ɑ-domain. Substrate docking into the active site of the GrTPS2 apo protein structure predicted catalytic amino acids. Biochemical characterization of protein variants identified residues with impact on enzyme activity and catalytic specificity. Specifically, mutagenesis of Y457 provided mechanistic insight into the position-specific deprotonation of the intermediary carbocation to form the characteristic 7,13 double bond of 7,13-copalyl diphosphate.

Functional characterization of a novel terpene synthase GaTPS1 involved in (E)-α-bergamotene biosynthesis in Gossypium arboreum

Terpenoids in plants are mainly synthesized by terpene synthases (TPSs), which play an important role in plant-environment interactions. Gossypium arboreum is one of the important cotton cultivars with excellent pest resistance, however, the biosynthesis of most terpenoids in this plant remains unknown. In this study, we performed a comparative transcriptome analysis of leaves from intact and Helicoverpa armigera-infested cotton plants. The results showed that the H. armigera infestation mainly induced the JA signaling pathway, ten TPS genes were differentially expressed in G. arboreum leaves. Among them, a novel terpene synthase, GaTPS1, was heterologously expressed and functionally characterized in vitro. The enzymatic reaction indicated that recombinant GaTPS1 was primarily responsible for the production of (E)-α-bergamotene. Moreover, molecular docking and site-directed mutagenesis analysis demonstrated that two amino acid residues, A412L and Y535F, distinctly influenced the catalytic activities and product specificity of GaTPS1. The mutants GaTPS1-A412L and GaTPS1-Y535F resulted in a decrease in the proportion of products (E)-α-bergamotene and D-limonene, while an increase in the proportion of products (E)-β-farnesene, α-pinene and β-myrcene. Our findings provide valuable insights into understanding the molecular basis of terpenoid diversity in G. arboreum, with potential applications in plant metabolism regulation and the improvement of resistant cotton cultivars.

Enzyme Machinery for Bacterial Glucoside Metabolism through a Conserved Non-hydrolytic Pathway.

The flexible acquisition of substrates from nutrient pools is critical for microbes to prevail in competitive environments. To acquire glucose from diverse glycoside and disaccharide substrates, many free-living and symbiotic bacteria have developed, alongside hydrolysis, a non-hydrolytic pathway comprised of four biochemical steps and conferred from a single glycoside utilization gene locus (GUL). Mechanistically, this pathway integrates within the framework of oxidation and reduction at the glucosyl/glucose C3, the eliminative cleavage of the glycosidic bond and the addition of water in two consecutive lyase-catalyzed reactions. Here, based on study of enzymes from the phytopathogen Agrobacterium tumefaciens, we reveal a conserved Mn2+ metallocenter active site in both lyases and identify the structural requirements for specific catalysis to elimination of 3-keto-glucosides and water addition to the resulting 2-hydroxy-3-keto-glycal product, yielding 3-keto-glucose. Extending our search of GUL-encoded putative lyases to the human gut commensal Bacteroides thetaiotaomicron, we discover a Ca2+ metallocenter active site in a putative glycoside hydrolase-like protein and demonstrate its catalytic function in the eliminative cleavage of 3-keto-glucosides of opposite (α) anomeric configuration as preferred by the A. tumefaciens enzyme (β). Structural and biochemical comparisons reveal the molecular-mechanistic origin of 3-keto-glucoside lyase stereo-complementarity. Our findings identify a basic set of GUL-encoded lyases for glucoside metabolism and assign physiological significance to GUL genetic diversity in the bacterial domain of life.

Enzymmaschinerie für bakteriellen Glucosidmetabolismus über einen konservierten nicht‐hydrolytischen Abbauweg

AbstractFlexible Substrataufnahme aus Nährstoffreservoirs ist für Mikroorganismen entscheidend, um sich in kompetitiven natürlichen Umgebungen durchzusetzen. Zur Aufnahme von Glucose aus verschiedenen Glucosid‐ und Disaccharid‐Substraten haben viele frei‐ und symbiotisch lebende Bakterien, neben der Hydrolyse, einen nicht‐hydrolytischen Stoffwechselweg entwickelt. Dieser besteht aus vier biochemischen Schritten und wird von einem distinkten Genlocus (glycoside utilization locus, GUL) codiert. Mechanistisch integriert der Stoffwechselweg die Oxidation und Reduktion am Glucosyl/Glucose‐C3 mit der eliminativen Spaltung der glycosidischen Bindung und der Addition von Wasser an das Eliminationsprodukt in zwei aufeinanderfolgenden, von Lyasen katalysierten Reaktionen. In dieser Studie zeigen wir, basierend auf der Untersuchung von Enzymen des Pflanzenpathogens Agrobacterium tumefaciens, ein konserviertes Aktivitätszentrum in beiden Lyasen, welches um ein zentrales Mn2+ Metallion angeordnet ist. Weiters identifizieren wir die strukturellen Voraussetzungen für die spezifische Katalyse der 3‐Ketoglucosid Eliminierung zu 2‐Hydroxy‐3‐ketoglycal und der Hydratisierung des Glycal zu 3‐Ketoglucose. Die Erweiterung unserer Suche nach GUL‐codierten putativen Lyasen auf das menschliche darmkommensale Bakterium Bacteroides thetaiotaomicron führte uns zur Entdeckung eines Ca2+‐koordinierten Aktivitätszentrums in einem Glycosidase‐ähnlichen Protein. Das Enzym katalysiert die eliminative Spaltung von 3‐Ketoglucosiden entgegengesetzter (α) anomerer Konfiguration im Vergleich zu den von der A. tumefaciens Lyase präferierten Substraten (β). Strukturelle und biochemische Vergleiche zeigen die molekular‐mechanistische Basis der Stereokomplementarität der beiden 3‐Ketoglucosid Lyasen. Unsere Ergebnisse identifizieren ein grundlegendes Set von GUL‐codierten Lyasen für den Glucosidmetabolismus und erlauben es, der Gendiversität von GULs in der Domäne von Bakterien physiologische Bedeutung zuzuschreiben.

Impact of metal oxides on the selectivity of NiBi/C catalysts for high-value C-3 products in glycerol electrooxidation reaction

Enhancing the catalytic efficiency and specificity of non-noble Nickel-based catalysts for glycerol electro-oxidation reaction (GEOR) is an ongoing challenge. Recently, metal oxides have attracted attention due to their unique physical properties, such as chemical stability at high anodic potentials, presence of oxygen vacancies, which can significantly influence reaction kinetics and pathways.In this study, we explored the impact of metal oxides CeO2, SnO2, and Sb2O3 • SnO2 (ATO) on the electrochemical performance and product selectivity of NiBi/C and Ni/C catalysts in glycerol electrooxidation (GEOR). The Ni/C-X and NiBi/C-X (X= CeO2, SnO2, and ATO) catalysts were synthesized through a sodium borohydride reduction method at room temperature and comprehensively characterized using various physiochemical and electrochemical techniques.Among the composite support catalysts, Ni and NiBi/C-X, Ni/C-ATO exhibited the highest peak current density, reaching 96 mA cm-2 at 1.50 V vs. RHE. To further assess the catalysts' performance, continuous electrolysis, coupled with HPLC analysis, was conducted to determine product selectivity. Notably, NiBi/C-ATO demonstrated remarkable selectivity, achieving 100 % lactate selectivity under mild conditions. These findings contribute valuable insights into optimizing non-noble Nickel-based catalysts for GEOR, particularly highlighting the promising performance of Ni/C-ATO in terms of selectivity towards valuable products. The DFT calculations provided insight into the synergistic interactions for GEOR over Ni-Ni and Bi-Bi nanoparticles.

Enzyme engineering in microbial biosynthesis of terpenoids: progress and perspectives

Terpenoids, known for their structural and functional diversity, are highly valued, especially in food, cosmetics, and cleaning products. Microbial biosynthesis has emerged as a sustainable and environmentally friendly approach for the production of terpenoids. However, the natural enzymes involved in the synthesis of terpenoids have problems such as low activity, poor specificity, and insufficient stability, which limit the biosynthesis efficiency. Enzyme engineering plays a pivotal role in the microbial synthesis of terpenoids. By modifying the structures and functions of key enzymes, researchers have significantly improved the catalytic activity, specificity, and stability of enzymes related to terpenoid synthesis, providing strong support for the sustainable production of terpenoids. This article reviews the strategies for the modification of key enzymes in microbial synthesis of terpenoids, including improving enzyme activity and stability, changing specificity, and promoting mass transfer through multi-enzyme collaboration. Additionally, this article looks forward to the challenges and development directions of enzyme engineering in the microbial synthesis of terpenoids.

Structural and functional analysis of plant ELO-like elongase for fatty acid elongation.

ELO-like elongase is a condensing enzyme elongating long chain fatty acids in eukaryotes. Eranthis hyemalis ELO-like elongase (EhELO1) is the first higher plant ELO-type elongase that is highly active in elongating a wide range of polyunsaturated fatty acids (PUFAs) and some monounsaturated fatty acids (MUFAs). This study attempted using domain swapping and site-directed mutagenesis of EhELO1 and EhELO2, a close homologue of EhELO1 but with no apparent elongase activity, to elucidate the structural determinants critical for catalytic activity and substrate specificity. Domain swapping analysis of the two showed that subdomain B in the C-terminal half of EhELO1 is essential for MUFA elongation while subdomain C in the C-terminal half of EhELO1 is essential for both PUFA and MUFA elongations, implying these regions are critical in defining the architecture of the substrate tunnel for substrate specificity. Site-directed mutagenesis showed that the glycine at position 220 in the subdomain C plays a key role in differentiating the function of the two elongases. In addition, valine at 161 and cysteine at 165 in subdomain A also play critical roles in defining the architecture of the deep substrate tunnel, thereby contributing significantly to the acceptance of, and interaction with primer substrates.

Characterization of switchgrass (Panicum virgatum L.) PvKSL1 as a levopimaradiene/abietadiene-type diterpene synthase.

The diverse class of plant diterpenoid metabolites serves important functions in mediating growth, chemical defence, and ecological adaptation. In major monocot crops, such as maize (Zea mays), rice (Oryza sativa), and barley (Hordeum vulgare), diterpenoids function as core components of biotic and abiotic stress resilience. Switchgrass (Panicum virgatum) is a perennial grass valued as a stress-resilient biofuel model crop. Previously we identified an unusually large diterpene synthase family that produces both common and species-specific diterpenoids, several of which accumulate in response to abiotic stress. Here, we report discovery and functional characterization of a previously unrecognized monofunctional class I diterpene synthase (PvKSL1) via in vivo co-expression assays with different copalyl pyrophosphate (CPP) isomers, structural and mutagenesis studies, as well as genomic and transcriptomic analyses. In particular, PvKSL1 converts ent-CPP into ent-abietadiene, ent-palustradiene, ent-levopimaradiene, and ent-neoabietadiene via a 13-hydroxy-8(14)-ent-abietene intermediate. Notably, although featuring a distinct ent-stereochemistry, this product profile is near-identical to bifunctional (+)-levopimaradiene/abietadiene synthases occurring in conifer trees. PvKSL1 has three of four active site residues previously shown to control (+)-levopimaradiene/abietadiene synthase catalytic specificity. However, mutagenesis studies suggest a distinct catalytic mechanism in PvKSL1. Genome localization of PvKSL1 distant from other diterpene synthases, and its phylogenetic distinctiveness from known abietane-forming diterpene synthases, support an independent evolution of PvKSL1 activity. Albeit at low levels, PvKSL1 gene expression predominantly in roots suggests a role of diterpenoid formation in belowground tissue. Together, these findings expand the known chemical and functional space of diterpenoid metabolism in monocot crops.

Nanozyme-Enabled Biomedical Diagnosis: Advances, Trends, and Challenges.

As nanoscale materials with the function of catalyzing substrates through enzymatic kinetics, nanozymes are regarded as potential alternatives to natural enzymes. Compared to protein-based enzymes, nanozymes exhibit attractive characteristics of low preparation cost, robust activity, flexible performance adjustment, and versatile functionalization. These advantages endow them with wide use from biochemical sensing and environmental remediation to medical theranostics. Especially in biomedical diagnosis, the feature of catalytic signal amplification provided by nanozymes makes them function as emerging labels for the detection of biomarkers and diseases, with rapid developments observed in recent years. To provide a comprehensive overview of recent progress made in this dynamic field, here an overview of biomedical diagnosis enabled by nanozymes is provided. This review first summarizes the synthesis of nanozyme materials and then discusses the main strategies applied to enhance their catalytic activity and specificity. Subsequently, representative utilization of nanozymes combined with biological elements in disease diagnosis is reviewed, including the detection of biomarkers related to metabolic, cardiovascular, nervous, and digestive diseases as well as cancers. Finally, some development trends in nanozyme-enabled biomedical diagnosis are highlighted, and corresponding challenges are also pointed out, aiming to inspire future efforts to further advance this promising field.

Preparation and application of single-atom nanozymes in oncology: a review.

Single-atom nanozymes (SAzymes) represent a cutting-edge advancement in nanomaterials, merging the high catalytic efficiency of natural enzymes with the benefits of atomic economy. Traditionally, natural enzymes exhibit high specificity and efficiency, but their stability are limited by environmental conditions and production costs. Here we show that SAzymes, with their large specific surface area and high atomic utilization, achieve superior catalytic activity. However, their high dispersibility poses stability challenges. Our review focuses on recent structural and preparative advancements aimed at enhancing the catalytic specificity and stability of SAzymes. Compared to previous nanozymes, SAzymes demonstrate significantly improved performance in biomedical applications, particularly in tumor medicine. This progress positions SAzymes as a promising tool for future cancer treatment strategies, integrating the robustness of inorganic materials with the specificity of biological systems. The development and application of SAzymes could revolutionize the field of biocatalysis, offering a stable, cost-effective alternative to natural enzymes.

Oligomerization of protein arginine methyltransferase 1 and its effect on methyltransferase activity and substrate specificity.

Proper protein arginine methylation by protein arginine methyltransferase 1 (PRMT1) is critical for maintaining cellular health, while dysregulation is often associated with disease. How the activity of PRMT1 is regulated is therefore paramount, but is not clearly understood. Several studies have observed higher order oligomeric species of PRMT1, but it is unclear if these exist at physiological concentrations and there is confusion in the literature about how oligomerization affects activity. We therefore sought to determine which oligomeric species of PRMT1 are physiologically relevant, and quantitatively correlate activity with specific oligomer forms. Through quantitative western blotting, we determined that concentrations of PRMT1 available in a variety of human cell lines are in the sub-micromolar to low micromolar range. Isothermal spectral shift binding data were modeled to a monomer/dimer/tetramer equilibrium with an EC50 for tetramer dissociation of ~20 nM. A combination of sedimentation velocity and Native polyacrylamide gel electrophoresis experiments directly confirmed that the major oligomeric species of PRMT1 at physiological concentrations would be dimers and tetramers. Surprisingly, the methyltransferase activity of a dimeric PRMT1 variant is similar to wild type, tetrameric PRMT1 with some purified substrates, but dimer and tetramer forms of PRMT1 show differences in catalytic efficiencies and substrate specificity for other substrates. Our results define an oligomerization paradigm for PRMT1, show that the biophysical characteristics of PRMT1 are poised to support a monomer/dimer/tetramer equilibrium in vivo, and suggest that the oligomeric state of PRMT1 could be used to regulate substrate specificity.

Identify Regioselective Residues of Ginsenoside Hydrolases by Graph-Based Active Learning from Molecular Dynamics.

Identifying the catalytic regioselectivity of enzymes remains a challenge. Compared to experimental trial-and-error approaches, computational methods like molecular dynamics simulations provide valuable insights into enzyme characteristics. However, the massive data generated by these simulations hinder the extraction of knowledge about enzyme catalytic mechanisms without adequate modeling techniques. Here, we propose a computational framework utilizing graph-based active learning from molecular dynamics to identify the regioselectivity of ginsenoside hydrolases (GHs), which selectively catalyze C6 or C20 positions to obtain rare deglycosylated bioactive compounds from Panax plants. Experimental results reveal that the dynamic-aware graph model can excellently distinguish GH regioselectivity with accuracy as high as 96-98% even when different enzyme-substrate systems exhibit similar dynamic behaviors. The active learning strategy equips our model to work robustly while reducing the reliance on dynamic data, indicating its capacity to mine sufficient knowledge from short multi-replica simulations. Moreover, the model's interpretability identified crucial residues and features associated with regioselectivity. Our findings contribute to the understanding of GH catalytic mechanisms and provide direct assistance for rational design to improve regioselectivity. We presented a general computational framework for modeling enzyme catalytic specificity from simulation data, paving the way for further integration of experimental and computational approaches in enzyme optimization and design.