Natural Substrate Research Articles

Deciphering the structure of a multi-drug resistant Acinetobacter baumannii short-chain dehydrogenase reductase.

The rapidly increasing threat of multi-drug-resistant Acinetobacter baumannii infections globally, encompassing a range of clinical manifestations from skin and soft tissue infections to life-threatening conditions like meningitis and pneumonia, underscores an urgent need for novel therapeutic strategies. These infections, prevalent in both hospital and community settings, present a formidable challenge to the healthcare system due to the bacterium's widespread nature and dwindling effective treatment options. Against this backdrop, the exploration of bacterial short-chain dehydrogenase reductases (SDRs) emerges as a promising avenue. These enzymes play pivotal roles in various critical bacterial processes, including fatty acid synthesis, homeostasis, metabolism, and contributing to drug resistance mechanisms. In this study, we present the first examination of the X-ray crystallographic structure of an uncharacterized SDR enzyme from A. baumannii. The tertiary structure of this SDR is distinguished by a central parallel β-sheet, consisting of seven strands, which is flanked by eight α-helices. This configuration exhibits structural parallels with other enzymes in the SDR family, underscoring a conserved architectural theme within this enzyme class. Despite the current ambiguity regarding the enzyme's natural substrate, the importance of many SDR enzymes as targets in anti-bacterial agent design is well-established. Therefore, the detailed structural insights provided in this study open new pathways for the in-silico design of therapeutic agents. By offering a structural blueprint, our findings may provide a platform for future research aimed at developing targeted treatments against this and other multi-drug-resistant infections.

Glyco-Engineering Cell Surfaces by Exo-Enzymatic Installation of GlcNAz and LacNAz Motifs.

Exo-enzymatic glyco-engineering of cell-surface glycoconjugates enables the selective display of well-defined glyco-motifs bearing bioorthogonal functional groups, which can be used to study glycans and their interactions with glycan-binding proteins. In recent years, strategies to edit cellular glycans by installing monosaccharides and their derivatives using glycosyltransferase enzymes have rapidly expanded. However, analogous methods to introduce chemical reporter-functionalized type 2 LacNAc motifs have not been reported. Herein, we report the chemo-enzymatic synthesis of unnatural UDP-GlcNAc and UDP-GalNAc nucleotide-sugars bearing azide, alkyne, and diazirine functionalities on the C2-acetamido group using the mutant uridylyltransferase AGX1F383A. The unnatural UDP-GlcNAc derivatives were examined as substrates for the human GlcNAc-transferase B3GNT2, where it was found that modified donors were tolerated for transfer, albeit to a lesser extent than the natural UDP-GlcNAc substrate. When the GlcNAc derivatives were examined as acceptor substrates for the human Gal-transferase B4GalT1, all derivatives were well tolerated and the enzyme could successfully form derivatized LacNAcs. B3GNT2 was also used to exo-enzymatically install GlcNAc and unnatural GlcNAc derivatives on cell-surface glycans. GlcNAc- or GlcNAz-engineered cells were further extended by B4GalT1 and UDP-Gal, producing LacNAc- or LacNAz-engineered cells. Our proof-of-concept glyco-engineering labeling strategy is amenable to different cell types and our work expands the exo-enzymatic glycan editing toolbox to selectively introduce unnatural type 2 LacNAc motifs.

Proteolytic, milk-clotting, and stability properties of proteases from Solanum elaeagnifolium fruit

Solanum elaeagnifolium (trompillo) has been considered a new alternative source of proteolytic enzymes for cheese-making. However, properties for this proteolytic source still need to be described. This study evaluated the proteolytic (PA) and milk-clotting activity (MCA) as well the stability properties for the proteases found in S. elaeagnifolium fruit extract (FE). Proteolytic enzymes were active in a broad pH range with a maximum at pH 10.0 and 80 °C and showed high stability in a wide pH (5.0–12.0) and temperature ranges (30–75 °C). MCA increased with the temperature in a range of 30–85 °C. Proteases from S. elaeagnifolium FE presented high thermostability, retaining 40% of the activity after 15 min of incubation at 80 °C. Temperatures higher than 85 °C caused the total PA loss. Phenylmethylsulphonyl fluoride (PMSF) inhibited more than 95% of the PA, indicating that the activity is mainly related to serine proteases. In addition, a casein hydrolysis profile, by SDS-PAGE, showed that proteases degrade α, β, and κ-casein with high affinity. S. elaeagnifolium fruit contains abundant proteolytic enzymes with high catalytic properties and activity over different protein substrates. Therefore, S. elaeagnifolium fruit represents a potential source of serine proteases for use in different biotechnological processes, offering attractive properties such as high activity in a broad pH and temperature range, high thermal stability, and preference over different natural substrates.

Unraveling the atomic mechanisms underlying glyphosate insensitivity in EPSPS: implications of distal mutations

5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS), as an indispensable enzyme in the shikimate pathway, is the specific target of grasser killer glyphosate (GPJ). GPJ is a competitive inhibitor of phosphoenolpyruvate (PEP), which is the natural substrate of EPSPS. A novel Ls-EPSPS gene variant discovered from Liliaceae, named ELs-EPSPS, includes five distal mutations, E112V, D142N, T351S, D425G, and R496G, endowing high GPJ insensitivity. However, the implicit molecular mechanism of the enhanced tolerance/insensitivity of GPJ in ELs-EPSPS is not fully understood. Herein, we try to interpret the hidden molecular mechanism using computational methods. Computational results reveal the enhanced flexibility of apo EPSPS upon mutations. The enhanced affinity of the initial binding substrate shikimate-3-phosphate (S3P), and the higher probability of second ligands PEP/GPJ entering the pocket are observed in the ELs-EPSPS-S3P system. Docking and MD results further confirmed the decreased GPJ-induced EPSPS inhibition upon mutations. And, the alterations of K98 and R179 side-chain orientations upon mutations are detrimental to GPJ binding at the active site. Additionally, the oscillation of side chain K98, in charge of PEP location, improves the proximity effect for substrates in the dual-substrate systems upon mutations. Our results clarify that the enhanced GPJ tolerance of EPSPS is achieved from decreased competitive inhibition of GPJ at the atomic perspective, and this finding further contributes to the cultivation of EPSPS genes with higher GPJ tolerance/insensitivity and a mighty renovation for developing glyphosate-resistant crops. Communicated by Ramaswamy H. Sarma

Ternary complexes of isopentenyl phosphate kinase from Thermococcus paralvinellae reveal molecular determinants of non-natural substrate specificity.

Isopentenyl phosphate kinases (IPKs) have recently garnered attention for their central role in biocatalytic "isoprenol pathways," which seek to reduce the synthesis of the isoprenoid precursors to two enzymatic steps. Furthermore, the natural promiscuity of IPKs toward non-natural alkyl-monophosphates (alkyl-Ps) as substrates has hinted at the isoprenol pathways' potential to access novel isoprenoids with potentially useful activities. However, only a handful of IPK crystal structures have been solved to date, and even fewer of these contain non-natural substrates bound in the active site. The current study sought to elucidate additional ternary complexes bound to non-natural substrates using the IPK homolog from Thermococcus paralvinellae (TcpIPK). Four such structures were solved, each bound to a different non-natural alkyl-P and the phosphoryl donor substrate/product adenosine triphosphate (ATP)/adenosine diphosphate (ADP). As expected, the quaternary, tertiary, and secondary structures of TcpIPK closely resembled those of IPKs published previously, and kinetic analysis of a novel alkyl-P substrate highlighted the potentially dramatic effects of altering the core scaffold of the natural substrate. Even more interesting, though, was the discovery of a trend correlating the position of two α helices in the active site with the magnitude of an IPK homolog's reaction rate for the natural reaction. Overall, the current structures of TcpIPK highlight the importance of continued structural analysis of the IPKs to better understand and optimize their activity with both natural and non-natural substrates.

Enhancement of nitrates removal in pilot-scale treatment wetland systems

ABSTRACT The primary objective of the present study is to enhance the denitrification process in constructed wetlands (CWs), employed as an ecological and natural technology for wastewater treatment. Eco-friendly approaches have been tested to improve the nitrate removal efficiency in pilot-scale subsurface flow CWs. Initially, the system’s performance was enhanced by using a natural substrate, namely cork, as a filter medium. Another approach is bio-inoculation into the rhizosphere of a bacterial strain with the potential capacity for nitrate removal, thereby reinforcing the synergy between the plant and microorganisms. Finally, the approaches were combined, and nitrate removal was assessed. The main results showed that the most effective nitrate removal from wastewater was achieved with cork (80%) compared to gravel (60%). Bio-inoculation into the rhizosphere of denitrifying bacteria significantly improved nitrate removal. Indeed, the nitrate removal rate by the inoculated CW filled with gravel, was higher than the non-inoculated system, with rates of 78% and 60%, respectively. Moreover, the highest removal rate of 99.77% was obtained with the inoculated system filled with cork.

Development and trends of constructed wetland substrates over the past 30 years: a literature visualization analysis based on CiteSpace.

Constructed wetland substrates (CWSs) have received considerable attention owing to their importance in adsorbing and degrading pollutants, providing growth attachment points for microorganisms, and supporting wetland plants. There are differences in the configurations and functions of constructed wetlands (CWs) for treating different water bodies and sewage, resulting in a wide variety of substrates. Research on the application and mechanism of CWSs is not sufficiently systematic. Therefore, the current research advancements and hotspots must be identified. Hence, we used CiteSpace to analyze 1955 English publications from the core collection database of the Web of Science to assess the current state of the CWS research field. Based on the cooperative network analysis, the roles of various countries, institutions, and authors in research on CWSs were reviewed. Keyword co-occurrence and cluster analyses were used to discuss the transformation of CWSs from removing traditional pollutants to emerging pollutants and the transition from incorporating natural substrates to artificial substrates. Finally, we underscored the need for more emphasis to be placed on the collocation and application of the CWSs at different latitudes. Furthermore, the substrate micro-interface process and its effects on the interaction patterns of pollutants and microorganisms should be thoroughly investigated to provide theoretical guidance for the development of wetland applications and mechanisms.

ISCOPEM2D V1.0: An In Situ Method to Characterize and Compare Chemical Vapor Deposition Graphene Films Using Quality Matrix Approaches

Single‐layer chemical vapor deposition (CVD) graphene films are commonly transferred from their native growth substrates during characterization. However, the transfer process influences their intrinsic material properties and growth substrate‐related material information is lost. Herein, an in situ method is presented to suspend CVD graphene films directly on their growth substrate. The approach makes the sample compatible for characterization across the widely used optical and electron microscopy techniques such as Raman spectroscopy and transmission electron microscopy. In addition, a quality matrix framework “ISCOPEM2D V1.0” is provided to support the quantification, comparison, and validation of various material properties of single‐layer graphene. The approach provides a holistic overview of various properties and facilitates quality control and reproducibility of single‐layer graphene‐based application development. Similar approaches can be useful to develop quality matrix‐based for other 2D materials.

Transport of metformin metabolites by guanidinium exporters of the small multidrug resistance family.

Proteins from the small multidrug resistance (SMR) family are frequently associated with horizontally transferred multidrug resistance gene arrays found in bacteria from wastewater and the human-adjacent biosphere. Recent studies suggest that a subset of SMR transporters might participate in the metabolism of the common pharmaceutical metformin by bacterial consortia. Here, we show that both genomic and plasmid-associated transporters of the SMRGdx functional subtype export byproducts of microbial metformin metabolism, with particularly high export efficiency for guanylurea. We use solid-supported membrane electrophysiology to evaluate the transport kinetics for guanylurea and native substrate guanidinium by four representative SMRGdx homologs. Using an internal reference to normalize independent electrophysiology experiments, we show that transport rates are comparable for genomic and plasmid-associated SMRGdx homologs, and using a proteoliposome-based transport assay, we show that 2 proton:1 substrate transport stoichiometry is maintained. Additional characterization of guanidinium and guanylurea export properties focuses on the structurally characterized homolog, Gdx-Clo, for which we examined the pH dependence and thermodynamics of substrate binding and solved an x-ray crystal structure with guanylurea bound. Together, these experiments contribute in two main ways. By providing the first detailed kinetic examination of the structurally characterized SMRGdx homolog Gdx-Clo, they provide a functional framework that will inform future mechanistic studies of this model transport protein. Second, this study casts light on a potential role for SMRGdx transporters in microbial handling of metformin and its microbial metabolic byproducts, providing insight into how native transport physiologies are co-opted to contend with new selective pressures.

Uncommon life history and winter spawning of common carp (Cyprinus carpio) in a natural thermal spring, under temperate climate.

Common carp female generally matures at age 4-5 years old and spawns between April and July under the temperate climate. Contrary to a range of 0-28 °C of temperate freshwaters, the water temperature of Lake Hévíz (Hungary, Central Europe), the largest natural bathable thermal lake in the world, varies between 26 and 35 °C seasonally. The specific environmental conditions (continuously warm water and its individual chemical composition, special nutrient base, lack of natural lakeside spawning substrate compared to usual spawning grounds, continuous high human disturbance, etc.) suggest that the carp population here may also differ in reproductive characteristics from their counterparts in surrounding waters. Our findings suggest that the self-sustaining dwarf common carp population of Lake Hévíz matures 2 to 4 years earlier (at the age of one) and spawns 1 to 3 months before (between February and April, at 27-30 °C water temperature) than carp typically do in the temperate zone (16-20 °C). Successful winter spawning was verified by rearing larvae from the collected eggs and in situ induced propagation.

Critical Challenges and Frontiers in Cartilage Tissue Engineering.

Cartilage tissue engineeringhas witnessed considerable advancements since its establishment in 1977, evolving from rudimentary surgical interventions to more nuanced biotechnological approaches. The field has navigated various challenges encompassing cellular considerations, scaffold material selection, environmental factors, and ethical and regulatory constraints. Innovations in cell source diversification, including chondrocytes, mesenchymal stem cells, and induced pluripotent stem cells, have been instrumental but not without their limitations, such as restricted cell proliferation and ethical dilemmas. Scaffold materials offer a unique dichotomy between natural substrates, which provide biocompatibility, and synthetic matrices, which grant mechanical integrity. However, translational hurdles in clinical applicability persist. Environmental factors, such as growth factors and thermal and mechanical forces, have been recognized as influential variables in cellular behavior and tissue maturation. Despite these strides, integration with host tissue remains a significant challenge, involving mechanical and immunological complexities. Looking forward, emerging technologies such as 3D and 4D printing, nanotechnology, and molecular therapies hold the promise of refining scaffold design and enhancing tissue regeneration. As the field continues to mature, a multidisciplinary approach encompassing thorough scientific investigation and collaboration is indispensable for overcoming existing challenges and realizing its full clinical potential.

Exploring a novel β-1,3-glucanosyltransglycosylase, MlGH17B, from a marine Muricauda lutaonensis strain for modification of laminari-oligosaccharides.

The marine environment, contains plentiful renewable resources, e.g. macroalgae with unique polysaccharides, motivating search for enzymes from marine microorganisms to explore conversion possibilities of the polysaccharides. In this study, the first GH17 glucanosyltransglycosylase, MlGH17B, from a marine bacterium (Muricauda lutaonensis), was characterized. The enzyme was moderately thermostable with Tm at 64.4°C and 73.2°C, but an activity optimum at 20°C, indicating temperature sensitive active site interactions. MlGH17B uses β-1,3 laminari-oligosaccharides with a degree of polymerization (DP) of 4 or higher as donors. Two glucose moieties (bound in the aglycone +1 and +2 subsites) are cleaved off from the reducing end of the donor while the remaining part (bound in the glycone subsites) is transferred to an incoming β-1,3 glucan acceptor, making a β-1,6-linkage, thereby synthesizing branched or kinked oligosaccharides. Synthesized oligosaccharides up to DP26 were detected by mass spectrometry analysis, showing that repeated transfer reactions occurred, resulting in several β-1,6-linked branches. The modeled structure revealed an active site comprising five subsites: three glycone (-3, -2 and -1) and two aglycone (+1 and +2) subsites, with significant conservation of substrate interactions compared to the only crystallized 1,3-β-glucanosyltransferase from GH17 (RmBgt17A from the compost thriving fungus Rhizomucor miehei), suggesting a common catalytic mechanism, despite different phylogenetic origin, growth environment, and natural substrate. Both enzymes lacked the subdomain extending the aglycone subsites, found in GH17 endo-β-glucanases from plants, but this extension was also missing in bacterial endoglucanases (modeled here), showing that this feature does not distinguish transglycosylation from hydrolysis, but may rather relate to phylogeny.

Investigation of natural compounds as methyltransferase inhibitors against dengue virus: an in silico approach

The global challenge posed by Dengue virus (DENV) infection persists, exacerbated by the absence of specific antiviral therapies. The viral methyltransferase (MTase) enzyme, crucial for viral RNA methylation and immune system evasion, has emerged as a promising drug target for combating Dengue fever. In this study, a comprehensive exploration of natural compounds derived from the COCONUT database was conducted, selecting 224 compounds based on their structural similarity to the native substrate of the MTase enzyme, S-adenosyl-L-methionine (SAM). Employing virtual screening techniques, four natural compounds (CNP0307160, CNP0082902, CNP0449158, and CNP0296775) with acceptable docking scores were selected for further re-docking after geometry optimization by the DFT method. Re-docking analyses unveiled significant interactions, including hydrogen bonds and hydrophobic interactions, between these selected ligands and the MTase protein. To gain deeper insights into the dynamic stability of these complexes, we conducted molecular dynamics simulations which showed lower RMSD values for CNP0307160, CNP0082902, and CNP0296775 when compared to the reference molecule. Furthermore, we assessed the structural and dynamic stability of the protein-ligand complexes through free binding energy calculations and Principal Component Analysis (PCA) of the simulation trajectories. In these analyses, the CNP0296775 compound exhibited promising results compared to the other three compounds. The cumulative findings of these investigations underscore the potential of CNP0296775 as a strong inhibitor of DENV MTase, thus offering a promising starting point for its further experimental validation and optimization. Communicated by Ramaswamy H. Sarma

Accumulation of polyethylene microplastics in river biofilms and effect on the uptake, biotransformation and toxicity of the antimicrobial triclosan

The interaction of multiple stressors in freshwater ecosystems may lead to adverse effects on aquatic communities and their ecological functions. Microplastics (MPs) are a class of contaminants of emerging concern that can exert both direct and indirect ecotoxicological effects. A growing number of studies have investigated MPs-attached microbial communities, but the interaction between MPs and substrate-associated biofilm (i.e., on natural river substrates, such as stones and sediments) remains poorly studied. In this work, the combined effects of polyethylene MPs (PE-MPs) with a particle size of 10–45 μm (2 mg/L) and the antimicrobial triclosan (TCS) (20 μg/L) were investigated on river biofilms through a short-term exposure experiment (72 h). To the best of authors’ knowledge, this is the first time that the combined effects of MPs and chemical contaminants in substrate-associated river biofilms were assessed. Different response parameters were evaluated, including (i) exposure assessment and ii) contaminants effects at different levels: bacterial community composition, antibiotic resistance, extracellular polymeric substances (EPS), photosynthetic efficiency (Yeff), and leucine aminopeptidase activity (LAPA). Triclosan was accumulated in river biofilms (1189–1513 ng/g dw) alongside its biotransformation product methyl-triclosan (20−29 ng/g dw). Also, PE-MPs were detected on biofilms (168−292 MP/cm2), but they had no significant influence on the bioaccumulation and biotransformation of TCS. A moderate shift in bacterial community composition was driven by TCS, regardless of PE-MPs co-exposure (e.g., increased relative abundance of Sphingomonadaceae family). Additionally, Yeff and EPS content were significantly disrupted in TCS-exposed biofilms. Therefore, the most remarkable effects on river biofilms were related to the antimicrobial TCS, whereas single PE-MPs exposure did not alter any of the evaluated parameters. These results demonstrate that biofilms might act as environmental sink of MPs. Although no interaction between PE-MPs and TCS was observed, the possible indirect impact of other MPs-adsorbed contaminants on biofilms should be further assessed.

Modern research on the dynamics of soil microbial communities in soils of Azerbaijan

In soils with minimal anthropogenic load the following were found B.cereus, B.muralis, B. Cibi, B. Simplex, Br.laterosporus, and in soils with high anthropogenic load the following were found B. cibi, B.cereus, P.lautus, B. pseudomycoides, B. niacini, B. mojavensis, B. agaradhaerens, B.arsenicus, B.firmus, B.licheniformis, B.atrophaeus, P. polymyxa, B.acidicola, B.drentensis, Br.laterosporus, B.subtilis. In anthropogenically transformed soils, the number of common bacilli species was reduced, while at each point species were found that were not found at other points. The proportion of microorganisms that utilize natural substrates is sharply reduced - in urban soils the number of aerobic spore-forming bacteria of the order Bacillales decreases several times.

New insights into pesticide occurrence and multicompartmental monitoring strategies in stream ecosystems using periphyton and suspended sediment

Streams are susceptible to pesticide pollutants which are transported outside of the intended area of application from surrounding agricultural fields. It is essential to monitor the occurrence and levels of pesticides in aquatic ecosystems to comprehend their effects on the aquatic environment. The common sampling strategy used for monitoring pesticides in stream ecosystems is through the collection and analysis of grab water samples. However, grab water sampling may not effectively monitor pesticides due to its limited ability to capture temporal and spatial variability, potentially missing fluctuations and uneven distribution of pesticides in aquatic environments. Monitoring using periphyton and sediment sampling may offer a more comprehensive approach by accounting for accumulative processes and temporal variations.Periphyton are a collective of microorganisms that grow on hard surfaces in aquatic ecosystems. They are responsive to chemical and biological changes in the environment, and therefore have the potential to act as a cost-effective, integrated sampling tool to monitor pesticide exposures in aquatic ecosystems. The objective of this study was to assess pesticides detected through periphyton, suspended sediment, and conventional grab water sampling methods and identify the matrix that offers a more comprehensive characterization of a stream's pesticide exposure profile. Ten streams across Southern Ontario were sampled in 2021 and 2022. At each stream site, water, sediment and periphyton, colonizing both artificial and natural substrates, were collected and analyzed for the presence of ~500 pesticides. Each of the three matrices detected distinctive pesticide exposure profiles. The frequency of detection in periphyton, sediment and water matrices were related to pesticides' log Kow and log Koc (P < 0.05). In addition, periphyton bioconcentrated 22 pesticides above levels observed in the ambient water. The bioconcentration factors of pesticides in periphyton can be predicted from their log Kow (simple linear regressions, P < 0.05). The results demonstrate that sediment and periphyton accumulate pesticides in stream environments. This highlights the importance of monitoring pesticide exposure using these matrices to ensure a complete and comprehensive characterization of exposure in stream ecosystems.

Coupling pathway prediction and fluorescence spectroscopy to assess the impact of auxiliary substrates on micropollutant biodegradation.

Some bacteria can degrade organic micropollutants (OMPs) as primary carbon sources. Due to typically low OMP concentrations, these bacteria may benefit from supplemental assimilation of natural substrates present in the pool of dissolved organic matter (DOM). The biodegradability of such auxiliary substrates and the impacts on OMP removal are tightly linked to biotransformation pathways. Here, we aimed to elucidate the biodegradability and effect of different DOM constituents for the carbofuran degrader Novosphingobium sp. KN65.2, using a novel approach that combines pathway prediction, laboratory experiments,and fluorescence spectroscopy. Pathway prediction suggested that ring hydroxylation reactions catalysed by Rieske-type dioxygenases and flavin-dependent monooxygenases determine the transformability of the 11 aromatic compounds used as model DOM constituents. Our approach further identified two groups with distinct transformation mechanisms amongst the four growth-supporting compounds selected for mixed substrate biodegradation experiments with the pesticide carbofuran (Group 1: 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde; Group 2: p-coumaric acid, ferulic acid). Carbofuran biodegradation kinetics were stable in the presence of both Group 1 and Group 2 auxiliary substrates. However, Group 2 substrates would be preferable for bioremediation processes, as they showed constant biodegradation kinetics under different experimental conditions (pre-growing KN65.2 on carbofuran vs. DOM constituent). Furthermore, Group 2 substrates were utilisable by KN65.2 in the presence of a competitor (Pseudomonas fluorescens sp. P17). Our study thus presents a simple and cost-efficient approach that reveals mechanistic insights into OMP-DOM biodegradation.

Revealing the mechanism of action of a first-in-class covalent inhibitor of KRASG12C (ON) and other functional properties of oncogenic KRAS by 31P NMR

Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient), and state 2 (active, effector binding enabled). Here we use 31P NMR to delineate the differences in state 1 and state 2 populations present in wild-type (WT) and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analogue GppNHp. Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in the state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RBD (RAS Binding Domain) binding and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small molecule inhibitor, BBO-8956, which is effective against both GDP and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs the state 1 - state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1 RBD is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action (MOA) for this novel, active-conformation inhibitor.

Synthetic Polymer Nanoparticles as an Abiotic Artificial Inhibitor of Tyrosinase.

An innovative methodology is presented for synthesizing synthetic polymer nanoparticles (TINPs) as potent tyrosinase inhibitors. This inhibition strategy combines the integration of two distinct functionalities, phenol, and phenylboronic acid, within the TINPs structure. The phenyl group mimics the natural monophenol substrate, forming a strong coordination with the catalytic copper ion, significantly inhibiting tyrosinase activity. Additionally, phenylboronic acid interacts with catechol, another tyrosinase substrate, further reducing enzyme efficiency. The shared benzene ring in phenyl and phenylboronic acid enhances binding to tyrosinase's hydrophobic pocket near its copper active site, contributing to potent inhibition. TINPs exhibit exceptional performance, boasting an impressive IC50 value of 3.5×10-8 m and an inhibition constant of 9.8×10-9 m. Validation of the approach is unequivocally demonstrated through the successful inhibition of tyrosinase activity and melanin production, substantiated in both in vitro and in vivo scenarios. The mechanism of TINP inhibition is elucidated through circular dichroism and Fourier transform infrared spectroscopy. This study introduces a versatile design approach for developing abiotic polymer-based enzyme inhibitors, expanding possibilities in enzyme inhibition research.

Evolution of Rhodopseudomonas palustris to degrade halogenated aromatic compounds involves changes in pathway regulation and enzyme specificity.

Halogenated aromatic compounds are used in a variety of industrial applications but can be harmful to humans and animals when released into the environment. Microorganisms that degrade halogenated aromatic compounds anaerobically have been isolated but the evolutionary path that they may have taken to acquire this ability is not well understood. A strain of the purple nonsulfur bacterium, Rhodopseudomonas palustris, RCB100, can use 3-chlorobenzoate (3-CBA) as a carbon source whereas a closely related strain, CGA009, cannot. To reconstruct the evolutionary events that enabled RCB100 to degrade 3-CBA, we isolated an evolved strain derived from CGA009 capable of growing on 3-CBA. Comparative whole-genome sequencing of the evolved strain and RCB100 revealed both strains contained large deletions encompassing badM, a transcriptional repressor of genes for anaerobic benzoate degradation. It was previously shown that in strain RCB100, a single nucleotide change in an alicyclic acid coenzyme A ligase gene, named aliA, gives rise to a variant AliA enzyme that has high activity with 3-CBA. When the RCB100 aliA allele and a deletion in badM were introduced into R. palustris CGA009, the resulting strain grew on 3-CBA at a similar rate as RCB100. This work provides an example of pathway evolution in which regulatory constraints were overcome to enable the selection of a variant of a promiscuous enzyme with enhanced substrate specificity.IMPORTANCEBiodegradation of man-made compounds often involves the activity of promiscuous enzymes whose native substrate is structurally similar to the man-made compound. Based on the enzymes involved, it is possible to predict what microorganisms are likely involved in biodegradation of anthropogenic compounds. However, there are examples of organisms that contain the required enzyme(s) and yet cannot metabolize these compounds. We found that even when the purple nonsulfur bacterium, Rhodopseudomonas palustris, encodes all the enzymes required for degradation of a halogenated aromatic compound, it is unable to metabolize that compound. Using adaptive evolution, we found that a regulatory mutation and a variant of promiscuous enzyme with increased substrate specificity were required. This work provides insight into how an environmental isolate evolved to use a halogenated aromatic compound.