Boron catalysis in a designer enzyme.

Enzymes play an increasingly important role in improving the benignity and efficiency of chemical production, yet the diversity of their applications lags heavily behind chemical catalysts as a result of the relatively narrow range of reaction mechanisms of enzymes. The creation of enzymes containing non-biological functionalities facilitates reaction mechanisms outside nature's canon and paves the way towards fully programmable biocatalysis1-3. Here we present a completely genetically encoded boronic-acid-containing designer enzyme with organocatalytic reactivity not achievable with natural or engineered biocatalysts4,5. This boron enzyme catalyses the kinetic resolution of hydroxyketones by oxime formation, in which crucial interactions with the protein scaffold assist in the catalysis. A directed evolution campaign led to a variant with natural-enzyme-like enantioselectivities for several different substrates. The unique activation mode of the boron enzyme was confirmed using X-ray crystallography, high-resolution mass spectrometry (HRMS) and 11B NMR spectroscopy. Our study demonstrates that genetic-code expansion can be used to create evolvable enantioselective enzymes that rely on xenobiotic catalytic moieties such as boronic acids and access reaction mechanisms not reachable through catalytic promiscuity of natural or engineered enzymes.

Redox Properties of Bacillus subtilis Ferredoxin:NADP+ Oxidoreductase: Potentiometric Characteristics and Reactions with Pro-Oxidant Xenobiotics.

Bacillus subtilis ferredoxin:NADP+ oxidoreductase (BsFNR) is a thioredoxin reductase-type FNR whose redox properties and reactivity with nonphysiological electron acceptors have been scarcely characterized. On the basis of redox reactions with 3-acetylpyridine adenine dinucleotide phosphate, the two-electron reduction midpoint potential of the flavin adenine dinucleotide (FAD) cofactor was estimated to be -0.240 V. Photoreduction using 5-deazaflavin mononucleotide (5-deazaFMN) as a photosensitizer revealed that the difference in the redox potentials between the first and second single-electron transfer steps was 0.024 V. We examined the mechanisms of the reduction of several different groups of non-physiological electron acceptors catalyzed by BsFNR. The reactivity of quinones and aromatic N-oxides toward BsFNR increased when increasing their single-electron reduction midpoint redox potentials. The reactivity of nitroaromatic compounds was lower due to their lower electron self-exchange rate, but it exhibited the same trend. A mixed single- and two-electron reduction reaction was characteristic of quinones, whereas reactions involving nitroaromatics proceeded exclusively via the one-electron reduction reaction. The oxidation of FADH• to FAD is the rate-limiting step during the oxidation of fully reduced FAD. The calculated electron transfer distances in the reaction with nitroaromatics were close to those of other FNRs including the plant-type enzymes, thus demonstrating their similar active site accessibility to low-molecular-weight oxidants despite the fundamental differences in their structures.

Costs of molecular adaptation to the chemical exposome: a focus on xenobiotic metabolism pathways.

Organisms adapt to their environment through different pathways. In vertebrates, xenobiotics are detected, metabolized and eliminated through the inducible xenobiotic-metabolizing pathways (XMP) which can also generate reactive toxic intermediates. In this review, we will discuss the impacts of the chemical exposome complexity on the balance between detoxication and side effects. There is a large discrepancy between the limited number of proteins involved in these pathways (few dozens) and the diversity and complexity of the chemical exposome (tens of thousands of chemicals). Several XMP proteins have a low specificity which allows them to bind and/or metabolize a large number of chemicals. This leads to undesired consequences, such as cross-inhibition, inefficient metabolism, release of toxic intermediates, etc. Furthermore, several XMP proteins have endogenous functions that may be disrupted upon exposure to exogenous chemicals. The gut microbiome produces a very large number of metabolites that enter the body and are part of the chemical exposome. It can metabolize xenobiotics and either eliminate them or lead to toxic derivatives. The complex interactions between chemicals of different origins will be illustrated by the diverse roles of the aryl hydrocarbon receptor which binds and transduces the signals of a large number of xenobiotics, microbiome metabolites, dietary chemicals and endogenous compounds. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

HGMMX: Host Gut Microbiota Metabolism Xenobiotics Database.

The metagenome of bacteria colonizing the human intestine is a set of genes that is almost 150 times greater than the set of host genes. Some of these genes encode enzymes whose functioning significantly expands the number of potential pathways for xenobiotic metabolism. The resulting metabolites can exhibit activity different from that of the parent compound. This can decrease the efficacy of pharmacotherapy as well as induce undesirable and potentially life-threatening side effects. Thus, analysis of the biotransformation of small drug-like compounds mediated by the gut microbiota is an important step in the development of new pharmaceutical agents and repurposing of the approved drugs. In vitro research, the interaction of drug-like compounds with the gut microbiota is a multistep and time-consuming process. Systematic testing of large sets of chemical structures is associated with a number of challenges, including the lack of standardized techniques and significant financial costs to identify the structure of the final metabolites. Estimation of the compounds' ability to be biotransformed by the gut microbiota and prediction of the structures of their metabolites are possible in silico. However, the development of computational approaches is limited by the lack of information about chemical structures metabolized by microbiota enzymes. The aim of this study is to create a database containing information on the metabolism of drug-like compounds by the gut microbiota. We created the data set containing information about 368 structures metabolized and 310 structures not metabolized by the human gut microbiota. The HGMMX database is freely available at https://www.way2drug.com/hgmmx. The information presented will be useful in the development of computational approaches for analyzing the impact of the human microbiota on metabolism of drug-like molecules.

VARIDT 2.0: structural variability of drug transporter.

The structural variability data of drug transporter (DT) are key for research on precision medicine and rational drug use. However, these valuable data are not sufficiently covered by the available databases. In this study, a major update of VARIDT (a database previously constructed to provide DTs' variability data) was thus described. First, the experimentally resolved structures of all DTs reported in the original VARIDT were discovered from PubMed and Protein Data Bank. Second, the structural variability data of each DT were collected by literature review, which included: (a) mutation-induced spatial variations in folded state, (b) difference among DT structures of human and model organisms, (c) outward/inward-facing DT conformations and (d) xenobiotics-driven alterations in the 3D complexes. Third, for those DTs without experimentally resolved structural variabilities, homology modeling was further applied as well-established protocol to enrich such valuable data. As a result, 145 mutation-induced spatial variations of 42 DTs, 1622 inter-species structures originating from 292 DTs, 118 outward/inward-facing conformations belonging to 59 DTs, and 822 xenobiotics-regulated structures in complex with 57 DTs were updated to VARIDT (https://idrblab.org/varidt/ and http://varidt.idrblab.net/). All in all, the newly collected structural variabilities will be indispensable for explaining drug sensitivity/selectivity, bridging preclinical research with clinical trial, revealing the mechanism underlying drug-drug interaction, and so on.

Concanavalin A: coordination diversity to xenobiotic metal ions and biological consequences.

The binding ability of lectins has gained attention owing to the carbohydrate-specific interactions of these proteins. Such interactions can be applied to diverse fields of biotechnology, including the detection, isolation, and concentration of biological target molecules. The physiological aspects of the lectin concanavalin A (ConA) have been intensively studied through structural and functional investigations. X-ray crystallography studies have proven that ConA has two ß-sheets and a short α-helix and that it exists in the form of a metalloprotein containing Mn2+ and Ca2+. These heterometals are coordinated with side chains located in a metal-coordinated domain (MCD), and they affect the structural environment in the carbohydrate-binding domain (CBD), which interacts with carbohydrates through hydrogen bonds. Recent studies have shown that ConA can regulate biophysical interactions with glycoproteins in virus envelopes because it specifically interacts with diverse polysaccharides through its CBD (Tyr, Asn, Asp, and Arg residues positioned next to the MCD). Owing to their protein-protein interaction abilities, ConA can form diverse self-assembled complexes including monomers, dimers, trimers, and tetramers, thus affording unique results in different applications. In this regard, herein, we present a review of the structural modifications in ConA through metal-ion coordination and their effect on complex formation. In recent approaches, ConA has been applied for viral protein detection, on the basis of the interactions of ConA. These aspects indicate that lectins should be thoroughly investigated with respect to their biophysical interactions, for avoiding unexpected changes in their interaction abilities.

An optimized method for RNA extraction from the polyurethane oligomer degrading strain Pseudomonas capeferrum TDA1 growing on aromatic substrates such as phenol and 2,4-diaminotoluene.

Bacterial degradation of xenobiotic compounds is an intense field of research already for decades. Lately, this research is complemented by downstream applications including Next Generation Sequencing (NGS), RT-PCR, qPCR, and RNA-seq. For most of these molecular applications, high-quality RNA is a fundamental necessity. However, during the degradation of aromatic substrates, phenolic or polyphenolic compounds such as polycatechols are formed and interact irreversibly with nucleic acids, making RNA extraction from these sources a major challenge. Therefore, we established a method for total RNA extraction from the aromatic degrading Pseudomonas capeferrum TDA1 based on RNAzol® RT, glycogen and a final cleaning step. It yields a high-quality RNA from cells grown on TDA1 and on phenol compared to standard assays conducted in the study. To our knowledge, this is the first report tackling the problem of polyphenolic compound interference with total RNA isolation in bacteria. It might be considered as a guideline to improve total RNA extraction from other bacterial species.

Biochemical aspects of hemoglobin-xenobiotic interactions and their implications in drug discovery.

Hemoglobin, a homodimeric globular protein, is found predominantly in red blood cells and in a small amount in blood plasma. Along with binding to certain native molecules, it also interacts with various xenobiotics. The present review aims at studying these interactions and the resultant tangible impact on the structure and function of the protein if any. The review also encompasses various analytical and computational approaches which are routinely used to study these interactions. A detailed discussion on types of interaction exhibited by individual xenobiotics has been included herein. Additionally, the effects of xenobiotic binding on the oxygen carrying capacity of hemoglobin have been reviewed. These insights would be of great value in drug design and discovery. Envisaging probable interactions of designed ligands with hemoglobin would help improvise the process of drug development. This would also open up new avenues for studying hemoglobin-mediated drug delivery.

A screening method for decoloration of xenobiotic dyes by fungi.

Wood degrading fungi are often screened for their ability to degrade xenobiotics such as dyes. Dye decoloration by these fungi on solid media could until now only be assessed qualitatively. We here describe a fast quantitative method to screen for dye decoloration on such media. Decoloration of crystal violet (CV), malachite green (MG), orange G (OG), rose bengal (RB) and remazol brilliant blue R (RBBR) by 124 isolates of the basidiomycete Schizophyllum commune was quantified with a flatbed scanner and the CIE-L*a*b* model. Colour and intensity changes were calculated with the Euclidean distance formula. More than 10 strains showed high MG decoloration. Isolates 136, 140 and 213 showed superior CV decoloration, while OG was most effectively decolorized by isolates 183, 216 and 227. Six strains showed high RB decoloration with isolate 216 being superior. The latter strain was also highly active on RBBR together with isolates 177 and 227. Together, dye decoloration was highly variable between the 124 isolates but strain 216 showed high activity on 3 out of 5 dyes. The fast screening method described in this paper enables identification of strains effectively decolorizing dyes.

Enzyme Kinetics of PAPS-Sulfotransferase.

The cytosolic sulfotransferase (SULT) enzymes are found in human liver, kidney, intestine, and other tissues. These enzymes catalyze the transfer of the -SO3 group from 3'-phospho-adenosyl-5'-phosphosulfate (PAPS) to a nucleophilic hydroxyl or amine group in a drug substrate. SULTs are stable as dimers, with a highly conserved dimerization domain near the C-terminus of the protein. Crystal structures have revealed flexible loop regions in the native proteins, one of which, located near the dimerization domain, is thought to form a gate that changes position once PAPS is bound to the PAPS-binding site and modulates substrate access and enzyme properties. There is also evidence that oxidation and reduction of certain cysteine residues reversibly regulate the binding of the substrate and PAPS or PAP to the enzyme thus modulating sulfonation. Because SULT enzymes have two substrates, the drug and PAPS, it is common to report apparent kinetic constants with either the drug or the PAPS varied while the other is kept at a constant concentration. The kinetics of product formation can follow classic Michaelis-Menten kinetics, typically over a narrow range of substrate concentrations. Over a wide range of substrate concentrations, it is common to observe partial or complete substrate inhibition with SULT enzymes. This chapter describes the function, tissue distribution, structural features, and properties of the human SULT enzymes and presents examples of enzyme kinetics with different substrates.

Massively parallel characterization of CYP2C9 variant enzyme activity and abundance.

CYP2C9 encodes a cytochrome P450 enzyme responsible for metabolizing up to 15% of small molecule drugs, and CYP2C9 variants can alter the safety and efficacy of these therapeutics. In particular, the anti-coagulant warfarin is prescribed to over 15 million people annually and polymorphisms in CYP2C9 can affect individual drug response and lead to an increased risk of hemorrhage. We developed click-seq, a pooled yeast-based activity assay, to test thousands of variants. Using click-seq, we measured the activity of 6,142 missense variants in yeast. We also measured the steady-state cellular abundance of 6,370 missense variants in a human cell line by using variant abundance by massively parallel sequencing (VAMP-seq). These data revealed that almost two-thirds of CYP2C9 variants showed decreased activity and that protein abundance accounted for half of the variation in CYP2C9 function. We also measured activity scores for 319 previously unannotated human variants, many of which may have clinical relevance.

Using QSAR Models to Predict Mitochondrial Targeting by Small-Molecule Xenobiotics Within Living Cells.

Prediction of mitochondrial targeting, or prediction of exclusion from mitochondria, of small-molecule xenobiotics (biocides, drugs, probes, toxins) can be achieved using an algorithm derived from QSAR modeling. Application of the algorithm requires knowing the chemical structures of all ionic species of the xenobiotic compound in question, and for certain numerical structure parameters (AI, CBN, log P, pK a, and Z) to be obtained for all such species. Procedures for specification of the chemical structures; estimation of the structure parameters; and application of the algorithm are described in an explicit protocol.

Electron Attachment to Isolated Molecules as a Probe to Understand Mitochondrial Reductive Processes.

This chapter describes the complementary experimental techniques Electron Transmission Spectroscopy and Dissociative Electron Attachment Spectroscopy, two of the most suitable means for investigating interactions between electrons and gas-phase molecules, resonance formation of temporary molecular negative ions, and their possible decay through the dissociative electron attachment (DEA) mechanism. The latter can be seen as the gas-phase counterpart of the transfer of a solvated electron in solution, accompanied by dissociation of the molecular anion, referred to as dissociative electron transfer (DET). DET takes place in vivo under reductive conditions, for instance, in the intermembrane space of mitochondria under interaction of xenobiotic molecules possessing high electron affinity with electrons "leaked" from the mitochondrial respiratory chain. A likely mechanism of the toxic activity of dichlorodiphenyltrichloroethane based on its DEA properties is briefly outlined, and compared with the well-established harmful effects of the model toxicant carbon tetrachloride ascribed to reductive dechlorination in a cellular ambient. A possible mechanism of the antioxidant activity of polyphenolic compounds present near the main site of superoxide anion production in mitochondria is also briefly discussed.

Mechanism-Based Inactivation of Cytochrome P450 Enzymes: Computational Insights.

Mechanism-based inactivation (MBI) refers to the metabolic bioactivation of a xenobiotic by cytochrome P450s to a highly reactive intermediate which subsequently binds to the enzyme and leads to the quasi-irreversible or irreversible inhibition. Xenobiotics, mainly drugs with specific functional units, are the major sources of MBI. Two possible consequences of MBI by medicinal compounds are drug-drug interaction and severe toxicity that are observed and highlighted by clinical experiments. Today almost all of these latent functional groups (e.g., thiophene, furan, alkylamines, etc.) are known, and their features and mechanisms of action, owing to the vast experimental and theoretical studies, are determined. In the past decade, molecular modeling techniques, mostly density functional theory, have revealed the most feasible mechanism that a drug undergoes by P450 enzymes to generate a highly reactive intermediate. In this review, we provide a comprehensive and detailed picture of computational advances toward the elucidation of the activation mechanisms of various known groups with MBI activity. To this aim, we briefly describe the computational concepts to carry out and analyze the mechanistic investigations, and then, we summarize the studies on compounds with known inhibition activity including thiophene, furan, alkylamines, terminal acetylene, etc. This study can be reference literature for both theoretical and experimental (bio)chemists in several different fields including rational drug design, the process of toxicity prevention, and the discovery of novel inhibitors and catalysts.

Human Family 1-4 cytochrome P450 enzymes involved in the metabolic activation of xenobiotic and physiological chemicals: an update.

This is an overview of the metabolic activation of drugs, natural products, physiological compounds, and general chemicals by the catalytic activity of cytochrome P450 enzymes belonging to Families 1-4. The data were collected from > 5152 references. The total number of data entries of reactions catalyzed by P450s Families 1-4 was 7696 of which 1121 (~ 15%) were defined as bioactivation reactions of different degrees. The data were divided into groups of General Chemicals, Drugs, Natural Products, and Physiological Compounds, presented in tabular form. The metabolism and bioactivation of selected examples of each group are discussed. In most of the cases, the metabolites are directly toxic chemicals reacting with cell macromolecules, but in some cases the metabolites formed are not direct toxicants but participate as substrates in succeeding metabolic reactions (e.g., conjugation reactions), the products of which are final toxicants. We identified a high level of activation for three groups of compounds (General Chemicals, Drugs, and Natural Products) yielding activated metabolites and the generally low participation of Physiological Compounds in bioactivation reactions. In the group of General Chemicals, P450 enzymes 1A1, 1A2, and 1B1 dominate in the formation of activated metabolites. Drugs are mostly activated by the enzyme P450 3A4, and Natural Products by P450s 1A2, 2E1, and 3A4. Physiological Compounds showed no clearly dominant enzyme, but the highest numbers of activations are attributed to P450 1A, 1B1, and 3A enzymes. The results thus show, perhaps not surprisingly, that Physiological Compounds are infrequent substrates in bioactivation reactions catalyzed by P450 enzyme Families 1-4, with the exception of estrogens and arachidonic acid. The results thus provide information on the enzymes that activate specific groups of chemicals to toxic metabolites.

A high-resolution MS/MS based strategy to improve xenobiotic metabolites analysis by metabolic pathway extension searching combined with parallel reaction monitoring: Flavonoid metabolism in wound site as a case.

For the analysis of xenobiotic metabolism, metabolites are commonly qualified by high-resolution mass spectrometry such as orbitrap or time-of-flight mass spectrometers, and quantified by triple-quadrupole (QQQ) mass spectrometer based multiple reaction monitoring. While this workflow shows drawback in the difficulty for instrumental parameters transfer, and QQQ provides less specificity. In this work, we constructed a high-resolution MS/MS (HR-MS/MS) based strategy to improve the discovery and quantification of unknown xenobiotic metabolites by metabolic pathway extension (MPE) searching combined with parallel reaction monitoring (PRM). Taking the flavonoid metabolism in diabetes wound S9 incubates as a test case. Firstly, MPE approach was used to screen all potential metabolites. In this step, an m/z value library of all theoretic flavonoid metabolites were constructed based on predefined flavonoid structures through 21 common xenobiotic metabolic reactions, and this library was matched with all features extracted from raw data (MS1 scan) of flavonoid-S9 co-incubate, then the matched features were exported into target list for MS2 fragmentation for structure validation. Secondly, the metabolites were relatively quantified by PRM mode based on their characteristic product ions. As a result, 131 metabolites of 9 different kinds of flavonoids in the skin and muscle were identified. To our best knowledge, this is the first report on the metabolism of flavonoids in the skin or muscle tissue. The results also validated the proposed HR-MS/MS-based strategy provided high specificity throughout both discovery and quantitation process of unknown xenobiotic metabolites without need of instrumental parameter transfer.

Mechanistic insights into the synergistic activation of the RXR-PXR heterodimer by endocrine disruptor mixtures.

Humans are chronically exposed to mixtures of xenobiotics referred to as endocrine-disrupting chemicals (EDCs). A vast body of literature links exposure to these chemicals with increased incidences of reproductive, metabolic, or neurological disorders. Moreover, recent data demonstrate that, when used in combination, chemicals have outcomes that cannot be predicted from their individual behavior. In its heterodimeric form with the retinoid X receptor (RXR), the pregnane X receptor (PXR) plays an essential role in controlling the mammalian xenobiotic response and mediates both beneficial and detrimental effects. Our previous work shed light on a mechanism by which a binary mixture of xenobiotics activates PXR in a synergistic fashion. Structural analysis revealed that mutual stabilization of the compounds within the ligand-binding pocket of PXR accounts for the enhancement of their binding affinity. In order to identify and characterize additional active mixtures, we combined a set of cell-based, biophysical, structural, and in vivo approaches. Our study reveals features that confirm the binding promiscuity of this receptor and its ability to accommodate bipartite ligands. We reveal previously unidentified binding mechanisms involving dynamic structural transitions and covalent coupling and report four binary mixtures eliciting graded synergistic activities. Last, we demonstrate that the robust activity obtained with two synergizing PXR ligands can be enhanced further in the presence of RXR environmental ligands. Our study reveals insights as to how low-dose EDC mixtures may alter physiology through interaction with RXR-PXR and potentially several other nuclear receptor heterodimers.

Comparative Toxicogenomics Database (CTD): update 2021.

The public Comparative Toxicogenomics Database (CTD; http://ctdbase.org/) is an innovative digital ecosystem that relates toxicological information for chemicals, genes, phenotypes, diseases, and exposures to advance understanding about human health. Literature-based, manually curated interactions are integrated to create a knowledgebase that harmonizes cross-species heterogeneous data for chemical exposures and their biological repercussions. In this biennial update, we report a 20% increase in CTD curated content and now provide 45 million toxicogenomic relationships for over 16 300 chemicals, 51 300 genes, 5500 phenotypes, 7200 diseases and 163 000 exposure events, from 600 comparative species. Furthermore, we increase the functionality of chemical-phenotype content with new data-tabs on CTD Disease pages (to help fill in knowledge gaps for environmental health) and new phenotype search parameters (for Batch Query and Venn analysis tools). As well, we introduce new CTD Anatomy pages that allow users to uniquely explore and analyze chemical-phenotype interactions from an anatomical perspective. Finally, we have enhanced CTD Chemical pages with new literature-based chemical synonyms (to improve querying) and added 1600 amino acid-based compounds (to increase chemical landscape). Together, these updates continue to augment CTD as a powerful resource for generating testable hypotheses about the etiologies and molecular mechanisms underlying environmentally influenced diseases.

GLORYx: Prediction of the Metabolites Resulting from Phase 1 and Phase 2 Biotransformations of Xenobiotics.

Predicting the structures of metabolites formed in humans can provide advantageous insights for the development of drugs and other compounds. Here we present GLORYx, which integrates machine learning-based site of metabolism (SoM) prediction with reaction rule sets to predict and rank the structures of metabolites that could potentially be formed by phase 1 and/or phase 2 metabolism. GLORYx extends the approach from our previously developed tool GLORY, which predicted metabolite structures for cytochrome P450-mediated metabolism only. A robust approach to ranking the predicted metabolites is attained by using the SoM probabilities predicted by the FAME 3 machine learning models to score the predicted metabolites. On a manually curated test data set containing both phase 1 and phase 2 metabolites, GLORYx achieves a recall of 77% and an area under the receiver operating characteristic curve (AUC) of 0.79. Separate analysis of performance on a large amount of freely available phase 1 and phase 2 metabolite data indicates that achieving a meaningful ranking of predicted metabolites is more difficult for phase 2 than for phase 1 metabolites. GLORYx is freely available as a web server at https://nerdd.zbh.uni-hamburg.de/ and is also provided as a software package upon request. The data sets as well as all the reaction rules from this work are also made freely available.

Global Substance Registration System: consistent scientific descriptions for substances related to health.

The US Food and Drug Administration (FDA) and the National Center for Advancing Translational Sciences (NCATS) have collaborated to publish rigorous scientific descriptions of substances relevant to regulated products. The FDA has adopted the global ISO 11238 data standard for the identification of substances in medicinal products and has populated a database to organize the agency's regulatory submissions and marketed products data. NCATS has worked with FDA to develop the Global Substance Registration System (GSRS) and produce a non-proprietary version of the database for public benefit. In 2019, more than half of all new drugs in clinical development were proteins, nucleic acid therapeutics, polymer products, structurally diverse natural products or cellular therapies. While multiple databases of small molecule chemical structures are available, this resource is unique in its application of regulatory standards for the identification of medicinal substances and its robust support for other substances in addition to small molecules. This public, manually curated dataset provides unique ingredient identifiers (UNIIs) and detailed descriptions for over 100 000 substances that are particularly relevant to medicine and translational research. The dataset can be accessed and queried at https://gsrs.ncats.nih.gov/app/substances.