Amino Acid Conjugates Research Articles

Dn-OPDA conjugation with amino acids inhibits its phytohormone bioactivity in Marchantia polymorpha.

Jasmonates are important phytohormones that regulate plant tolerance to biotic and abiotic stresses, and developmental processes. Distinct jasmonates in different plant lineages activate a conserved signalling pathway that mediates these responses: dinor-12-oxo-phytodienoic acid (dn-OPDA) isomers in bryophytes and lycophytes, and JA-Ile in most vascular plants. In many cases, the final responses triggered by these phytohormones depend on the accumulation of specialized metabolites. To identify compounds regulated by the dn-OPDA pathway in the liverwort Marchantia polymorpha, untargeted metabolomic analyses were carried out in response to wounding, a stress that activates the dn-OPDA pathway. A previously unreported group of molecules was identified from these analyses: dn-OPDA-amino acid conjugates (dn-OPDA-aas). Their accumulation after wounding and herbivory was confirmed by targeted metabolic profiling in Marchantia and in all species in which we previously detected dn-iso-OPDA. Mutants in GRETCHEN-HAGEN 3A (MpGH3A) failed to accumulate dn-OPDA-aa conjugates and showed a constitutive activation of the OPDA pathway and increased resistance to herbivory. Our results show that dn-iso-OPDA bioactivity is reduced by amino acid conjugation. Therefore, jasmonate conjugation in land plants plays dichotomous roles: jasmonic acid (JA) conjugation with isoleucine (Ile) produces the bioactive JA-Ile in tracheophytes, whereas conjugation of dn-iso-OPDA with different amino acids deactivates the phytohormone in bryophytes and lycophytes.

Light-Activated Reactivity of Nitrones with Amino Acids and Proteins.

Controlled modifications of amino acids are an indispensable tool for advancing fundamental and translational research based on peptides and proteins. Yet, we still lack methods to chemically modify each naturally occurring amino acid sidechain. To help address this gap, we show that N,α-diaryl oxaziridines expand the scope of bioconjugation methods to chemically modify cysteine, methionine, and tryptophan residues with evidence for additional tyrosine labelling in a proteomic context. Conjugation primarily at tryptophan sites can be accessed by selective cleavage of modifications at other sidechains. The N,α-diaryl oxaziridine reagents are accessed through photoisomerization of nitrones, which serve as photocaged reagents, thus providing an additional level of control over reactivity. Initial guiding principles for the design of nitrone reagents are developed by exploring the impact of structure on UV-vis absorption, photoisomerization, and reactivity. We identify a nitrone structure that maximizes photoisomerization efficiency, the aqueous stability of the oxaziridine, the extent of amino acid modification, and the stability of the resulting amino acid conjugates. We then translate nitrone reagents to modify proteins in aqueous conditions. Finally, we use nitrones to profile reactive residues across the proteome of a mammalian cell line and find that they expand the proteome coverage.

Structure-Dependent uptake and metabolism of Tire additives Benzothiazoles in carrot plant

Tire additives, such as benzothiazole and its derivatives (collectively called BTs), are large-volume chemicals that are constantly emitted into agricultural environment via tire-road wearing and other actions. The potential accumulation of BTs in food crops depends largely on their metabolism in plants, which is poorly understood. Herein, we evaluated uptake and metabolism of six BTs in carrot callus and intact carrot plants to understand their structure-specific metabolism. All BTs were readily taken up by carrot roots, with their root concentration factors (RCF) ranging from 1.66 ± 0.01 to 2.95 ± 0.05. Although the tested BTs exhibited poor upward translocation from root to leaves (translocation factors < 1), the translocation factors of 2-methylbenzothiazole (0.79) and 2-aminobenzothiazole (0.65) were significantly higher than that of 2-methylbenzothiazole (0.18) and 2-(methylthio)benzothiazole (0.22). These results indicated the structure-dependent uptake and translocation of BTs in carrot. Correlation analysis between log Kow and log RCF or TF revealed that the hydrophobicity of BTs predominantly affected their root uptake and acropetal translocation in carrots. With the aid of high-resolution mass spectrometry, a total of 18 novel metabolites of BTs were tentatively identified, suggesting that BT compounds can be metabolized by carrot callus. The proposed metabolites of BTs include four hydroxylated products, one demethylated product, five glycosylated products and eight amino acid conjugated products, revealing that glycosylation and amino acid conjugation were the dominant transformation pathways for BT metabolism in carrot. However, the detected species of metabolites for six BTs varied distinctly, indicating structure-specific metabolism of BTs in plants. The findings of this study improve our understanding of structure-dependent fate and transformation of BTs in plants. Since BTs metabolites in food crops could present an unintended exposure route to consumers, the structure-specific differences of BTs uptake, metabolism and accumulation in plants must be considered when addressing human dietary exposure risks.

Amino Acid–Based Block Copolymer Templates‐Dependent Enhancement of Physicochemical and Electrochemical Characteristics of Mesoporous Carbons

ABSTRACTAmino acid–based block copolymers (BCPs) have distinct features such as secondary structure formation, chirality, amphoteric nature, non‐toxicity, and biodegradability, setting them apart from other BCPs. This suggests that amino acid–based BCPs may exhibit unique self‐assembly behaviors. Despite these advantages, they have not yet been utilized as templates for mesoporous carbons (MCs) synthesis. Here, we investigate, for the first time, the effect of hydrophobicity of two different poly(ethylene glycol) (PEG) conjugated amino acid–based BCP templates (PEG‐poly(β‐benzyl‐l‐aspartate) (PEG‐PBLA) and PEG‐poly(γ‐benzyl‐l‐glutamate) (PEG‐PBLG)) on the construction of MC materials and also their physicochemical and electrochemical characteristics. MCs produced using PEG‐PBLA and PEG‐PBLG as templates are labeled CPBLA and CPBLG, respectively. Utilizing amino acid–based BCP systems enabled achieving MC materials with nearly 1 at% nitrogen doping without external nitrogen dopants. Physicochemical analysis showed CPBLA had a smaller particle size, higher specific surface area, pore size, and hydrophilicity due to increased oxygen and nitrogen contents, as well as a higher defective structure than CPBLG. The higher hydrophilicity of PEG‐PBLA led to the formation of CPBLA MC particles with smaller size and higher specific surface area of 602 m2g−1 and pore size of 7.8 nm. Cyclic voltammetry demonstrated that CPBLA had superior charge‐storing capacity (specific capacitance of 147 F g−1 at 1 mv s−1) than CPBLG, attributed to its better physicochemical properties. These promising findings suggest that amino acid–based BCP systems can serve not only as templates but also as carbon and nitrogen sources, offering potential for high‐performance electrochemical energy storage devices.

Ultrasound-Assisted Solid-Phase Affibody Synthesis Using ZEGFR:1907 as an Example-Superior to the Conventional Protocol?

Affibody molecules represent a class of highly specific binders of particular interest for the development of highly affine target-specific radiopharmaceuticals. Their chemical synthesis is, however, intricate due to their considerable length of 58 amino acids; thus, approaches to optimize their preparation are constantly being sought. As ultrasound assistance has recently been shown to increase the efficiency of amino acid conjugation during solid-phase peptide synthesis (SPPS), the influence of ultrasonication on the outcome of the SPPS-based preparation of the EGFR-specific affibody ZEGFR:1907 was compared to a common protocol relying on mechanical shaking. After the identification of a suitable solid support for the study, the execution of the systematic comparison of both approaches showed that conventional and ultrasound-assisted syntheses yielded equivalent results with analogous composition of the raw products. Further, both approaches produced the affibody in good isolated yields of >20% when applying the same optimal reagent excesses and coupling times for the conjugation of each amino acid. This indicates that, under optimal reaction conditions, the choice of solid support used has a much stronger influence on the outcome of the preparation of ZEGFR:1907 than the application of ultrasound, which did not further improve the synthesis results. Therefore, for the chemical synthesis of affibodies, great attention should be paid to the choice of a suitable solid support, enabling this highly interesting class of biomolecules to be obtained in good yields and to bring them more into the focus of radiopharmaceutical research.

A secondary β-hydroxybutyrate metabolic pathway linked to energy balance.

β-hydroxybutyrate (BHB) is an abundant ketone body. To date, all known pathways of BHB metabolism involve interconversion of BHB and primary energy intermediates. Here we show that CNDP2 controls a previously undescribed secondary BHB metabolic pathway via enzymatic conjugation of BHB and free amino acids. This BHB-ylation reaction produces a family of endogenous ketone metabolites, the BHB-amino acids. Genetic ablation of CNDP2 in mice eliminates tissue amino acid BHB-ylation activity and reduces BHB-amino acid levels. Administration of BHB-Phe, the most abundant BHB-amino acid, to obese mice activates neural populations in the hypothalamus and brainstem and suppresses feeding and body weight. Conversely, CNDP2-KO mice exhibit increased food intake and body weight upon ketosis stimuli. CNDP2-dependent amino acid BHB-ylation and BHB-amino acid metabolites are also conserved in humans. Therefore, the metabolic pathways of BHB extend beyond primary metabolism and include secondary ketone metabolites linked to energy balance.

Origami of KR-12 Designed Antimicrobial Peptides and Their Potential Applications.

This review describes the discovery, structure, activity, engineered constructs, and applications of KR-12, the smallest antibacterial peptide of human cathelicidin LL-37, the production of which can be induced under sunlight or by vitamin D. It is a moonlighting peptide that shows both antimicrobial and immune-regulatory effects. Compared to LL-37, KR-12 is extremely appealing due to its small size, lack of toxicity, and narrow-spectrum antimicrobial activity. Consequently, various KR-12 peptides have been engineered to tune peptide activity and stability via amino acid substitution, end capping, hybridization, conjugation, sidechain stapling, and backbone macrocyclization. We also mention recently discovered peptides KR-8 and RIK-10 that are shorter than KR-12. Nano-formulation provides an avenue to targeted delivery, controlled release, and increased bioavailability. In addition, KR-12 has been covalently immobilized on biomaterials/medical implants to prevent biofilm formation. These constructs with enhanced potency and stability are demonstrated to eradicate drug-resistant pathogens, disrupt preformed biofilms, neutralize endotoxins, and regulate host immune responses. Also highlighted are the safety and efficacy of these peptides in various topical and systemic animal models. Finaly, we summarize the achievements and discuss future developments of KR-12 peptides as cosmetic preservatives, novel antibiotics, anti-inflammatory peptides, and microbiota-restoring agents.

Single-Walled Carbon Nanotubes As Fluorescent Probes for Supramolecular Self-Assembly Complexes

Semiconducting single-walled carbon nanotubes (SWCNTs) are distinguished by their fluorescence in the near-infrared range, which overlaps with the transparency window of biological samples, and they do not photobleach or blink. Utilizing their unique optical properties, SWCNTs can be rendered optical probes for the characterization and analysis of bio-inspired peptide-based hydrogels. Peptide hydrogels, formed through the supramolecular self-assembly of conjugated amino acids, provide versatile three-dimensional scaffolds for applications in cell culture, tissue engineering, and regenerative medicine. The inherent properties of these amino acids or peptides, notably their limited solubility in water, drive them to self-assemble into elongated fibrillary nanostructures, ultimately creating self-supporting hydrogel matrices. We integrate near-infrared fluorescent SWCNTs into these peptide hydrogels as dynamic fluorescent probes, offering real-time insights into the morphological and structural evolution of the hydrogels. This approach not only enhances our understanding of the self-assembly mechanisms at play but also opens new avenues in supramolecular science. The integration of SWCNTs into the supramolecular structures showcases their potential as a powerful tool for long-term, non-invasive monitoring, with immense promise for numerous biomedical applications.

24-epibrassinolide as a multidimensional regulator of rice (Oryza sativa) physiological and molecular responses under isoproturon stress

Brassinosteroids (BRs) can regulate various processes in plant development and defense against environmental stress. In this study, the contribution of BRs in the degradation of isoproturon (IPU) in rice has been established. IPU has a significant effect on rice growth, chlorophyll content, and membrane permeability. When treated with 1.0 μmol/L 24-epibrassinolide (EBR), a BR analogue, the associated symptoms of rice poisoning were alleviated as the IPU levels in the rice and growth media were decreased. In the presence of EBR, the activities of several IPU-related detoxification enzymes were enhanced to cope with the stress due to IPU. An RNA-sequencing (RNA-Seq) has been performed to determine the variation of transcriptomes and metabolic mechanisms in rice treated with EBR, IPU, or IPU+EBR. Some of the differentially expressed genes (DEGs) were Phase I-III reaction components of plants, such as cytochrome P450 (CYP450), glutathione S-transferase (GST), glycosyltransferases (GTs), and the ATP-binding cassette transporter (ABC transporter). The expression of some signal transduction genes was significantly up-regulated. The relative content of low-toxicity IPU metabolites increased due to the presence of EBR as determined by UPLC/Q-TOF-MS/MS. The IPU metabolic pathways include enzyme-catalyzed demethylation, hydroxylation, hydrolysis, glycosylation, and amino acid conjugation processes. The results suggest that EBR plays a key role in the degradation and detoxification of IPU. This study has provided evidence that BRs regulate the metabolism and detoxification of IPU in rice, and offers a new approach to ensuring cleaner crops by eliminating pesticide residues in the environment.

Knock-Out of ACY-1 Like Gene in Spodoptera litura Supports the Notion that FACs Improve Nitrogen Metabolism.

Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and N-linolenoyl-L-glutamine were originally identified in the regurgitant of Spodoptera exigua larvae. These fatty acid amino acid conjugates (FACs) are known to be elicitors that induce plants to release volatile compounds which in turn attract natural enemies of the larvae such as parasitic wasps. FAC concentrations are regulated by enzymatic biosynthesis and hydrolysis in the intestine of Lepidoptera larvae. It has been proposed that FAC metabolism activates glutamine synthetase and plays an important role in nitrogen metabolism in larvae. In this study, we identified candidate genes encoding a FACs hydrolase in Spodoptera litura using genomic information of various related lepidopteran species in which FACs hydrolases have been reported. We analyzed the importance of FAC hydrolysis on caterpillar performance with CRISPR/Cas9 knock outs. Larvae of strains with an inactive FACs hydrolase excreted FACs in their feces. They absorbed 30% less nitrogen from the diet compared to WT caterpillars resulting in a reduction of their body weight of up to 40% compared to wild type caterpillars. These results suggest that the hydrolysis of FACs is an important metabolism for insects and that FACs are important for larval growth.

Comparative profiling of serum, urine, and feces bile acids in humans, rats, and mice.

Bile acids (BAs) play important pathophysiological roles in both humans and mammalian animals. Laboratory rats and mice are widely used animal models for assessing pharmacological effects and their underlying molecular mechanisms. However, substantial physiological differences exist in BA composition between humans and murine rodents. Here, we comprehensively compare BA profiles, including primary and secondary BAs, along with their amino acid conjugates, and sulfated metabolites in serum, urine, and feces between humans and two murine rodents. We further analyze the capabilities in gut microbial transform BAs among three species and compare sex-dependent variations within each species. As a result, BAs undergo amidation predominately with glycine in humans and taurine in mice but are primarily unamidated in rats. BA sulfation is a unique characteristic in humans, whereas rats and mice primarily perform multiple hydroxylations during BA synthesis and metabolism. For gut microbial transformed BA capabilities, humans are comparable to those of rats, stronger than those of mice in deconjugation and 7α-dehydroxylation, while humans are weak than those of rats or mice in oxidation and epimerization. Such differences enhance our understanding of the divergent experimental outcomes observed in humans and murine rodents, necessitating caution when translating findings from these rodent species to humans.

Sustainable production of multifunctional melanin-like amino acid surfactant by conjugating o-quinone building block from natural polyphenols

Surfactants occupy a prominent place in the domestic and industrial fields. Amino acid surfactants have attracted attention as alternatives to petrochemical-based surfactants owing to their good biocompatibility and biodegradability. Conventional amino acid surfactants with structures having only acyl linkages lack multifunctionality. Therefore, novel amino acid surfactants must be developed to achieve this feature. Inspired by melanogenesis, the biomimetic synthesis of melanin-like amino acid surfactants with structures similar to that of melanin was conceived. Natural polyphenols with catechol moieties serve as building blocks for hydrophobic moieties. As a proof-of-concept, caffeic acid phenethyl ester was oxidized to o-quinone using periodate resin. Melanin-like amino acid surfactants were synthesized by conjugating amino acids to o-quinones via Michael addition and Schiff base reactions. The structure was confirmed by mass spectrometry. Melanin-like amino acid surfactants have outstanding surface activity with a low critical micelle concentration and excellent capacity for foaming, emulsifying, dispersing, wetting, and solubilizing. Melanin-like amino acid surfactants exhibit low skin irritation, as well as high tolerance to ions and pH. The utilization of polyphenols introduces multifunctionality, affording antioxidant, antibacterial and whitening activities. This study proposes a modular strategy for fabricating new and multifunctional amino acid surfactants that mimic melanin by simply replacing the o-quinone building block. This biomimetic synthesis route is a sustainable process for the green production of amino acid surfactants. This new generation of amino acid surfactants will contribute to meeting the increasing demand for multifunctional surfactants in both academia and industry.

Metabolomics and amino acid profiling of plasma reveals the metabolite profiles associated with nitrogen utilisation efficiency in primiparous dairy cows

Nitrogen (N) utilisation efficiency (NUE, milk N yield [g/d]/N intake [g/d]) is an important performance indicator in dairy farming. Determining the NUE-associated blood metabolite profile will contribute to the optimisation of nutritional strategies to further improve NUE among dairy cows. Here, 20 primiparous lactating cows with days in milk ranging from 95 to 115 days were selected from a total of 1 221 cows. Each cow’s N intake and milk N yield were measured for 7 days. Subsequently, blood samples were collected before morning feeding. Based on analysis and calculations, cows were retrospectively classified into two groups based on their NUE values, namely, a low NUE group (LNUE, NUE = 24.8 ± 1.6%, n = 10, mean ± SD) and a high NUE group (HNUE, NUE = 35.2 ± 1.7%, n = 10, mean ± SD). Plasma samples were selected from six cows in each group for metabolomics and amino acid profiling. Among the 41 differential metabolites (DMs) identified in the metabolomic analysis, sucrose, MG(0:0/22:1(13Z)/0:0), 2-amino-6-hydroxyhexanoic acid, and L-glutamine exhibited significant correlations with NUE, milk yield, and BW (P < 0.05). Moreover, the five differential amino acids and amino acid metabolites (DAAs) identified in the amino acid profiling and 5 of the 6 differential amino acids and amino acid conjugates identified by plasma metabolomics were found to be less abundant in the HNUE group (P < 0.05). Specifically, there was a 39.4% decrease in L-arginine content and a 29.2% decrease in L-glutamine content (P < 0.05). Pathway analysis indicated that the DMs and DAAs were mainly involved in arginine biosynthesis, glutathione metabolism, arginine and proline metabolism, and tryptophan metabolism (pathway impact > 0.1). These results provided new insights into the new blood metabolite profile associated with NUE in dairy cows. These new insights can provide foundational information for the formulation of new strategies to further enhance NUE in dairy cows.

Single-walled carbon nanotubes as near-infrared fluorescent probes for bio-inspired supramolecular self-assembled hydrogels

Hydrogels derived from fluorenylmethoxycarbonyl (Fmoc)-conjugated amino acids and peptides demonstrate remarkable potential in biomedical applications, including drug delivery, tissue regeneration, and tissue engineering. These hydrogels can be injectable, offering a minimally invasive approach to hydrogel implantation. Given their potential for prolonged application, there is a need for non-destructive evaluation of their properties over extended periods. Thus, we introduce a hydrogel characterization platform employing single-walled carbon nanotubes (SWCNTs) as near-infrared (NIR) fluorescent probes. Our approach involves generating supramolecular self-assembling hydrogels from aromatic Fmoc-amino acids. Integrating SWCNTs into the hydrogels maintains their structural and mechanical properties, establishing SWCNTs as optical probes for hydrogels. We demonstrate that the SWCNT NIR-fluorescence changes during the gelation process correlate to rheological changes within the hydrogels. Additionally, single particle tracking of SWCNTs incorporated in the hydrogels provides insights into differences in hydrogel morphologies. Furthermore, the disassembly process of the hydrogels can be monitored through the SWCNT fluorescence modulation. The unique attribute of SWCNTs as non-photobleaching fluorescent sensors, emitting at the biologically transparent window, offers a non-destructive method for studying hydrogel dynamics over extended periods. This platform could be applied to a wide range of self-assembling hydrogels to advance our understanding and applications of supramolecular assembly technologies.

The GH3 Protein from the Clubroot Pathogen Plasmodiophora brassicae Causes Hormone-Related Phenotypes in Arabidopsis thaliana

In the genome of the obligate biotrophic protist Plasmodiophora brassicae, which causes clubroot disease in the Brassicaceae family, a single gene was identified with homology to the group of GH3 proteins synthesizing amino acid conjugates with carboxylic acids. In comparison to plant members the P. brassicae GH3 protein seems to be very promiscuous with respect to the substrates converted. In vitro assays showed conversion of three different auxins (indole-3-acetic, IAA; indole-propionic, IPA; and indole-3-butyric acids, IBA), jasmonic acid (JA) and also 12-oxo-phytodienoic acid (OPDA). Auxin and Methyl-JA responsive elements were found in the promoter sequence of PbGH3. For further analysis Arabidopsis thaliana was transformed with PbGH3. Seedlings with confirmed constitutive PbGH3 protein production were grown to adult plants that were shown to produce fewer inflorescences per plant. Growth on 10 µM IAA or JA levels also altered the phenotype of the seedlings, but not so much between wild types and transgenic lines. The hormone profiles in seedlings and adult plants were examined for IAA, JA, OPDA, and respective conjugates. Transgenic seedlings displayed changes that could be partially attributed to the overexpression of a GH3 gene like lower levels of free IAA and the JA precursor cis-OPDA under control conditions, and higher levels of some auxin conjugates accumulated after IAA treatment compared to the corresponding wild types. However, the hormone patterns in adult plants were more complex and varied based on different tissue types analyzed. After inoculation with P. brassicae the transgenic plants did not show any changes in infection rate and disease index, but had higher shoot and lower root weight in infected plants compared to controls. Based on the above findings we discuss a role for PbGH3 during pathogenesis.

Genome Mining of a Fungal Polyketide Synthase-Nonribosomal Peptide Synthetase Hybrid Megasynthetase Pathway to Synthesize a Phytotoxic N-Acyl Amino Acid.

Guided by the retrobiosynthesis hypothesis, we characterized a fungal polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) hybrid megasynthetase pathway to generate 2-trans-4-trans-2-methylsorbyl-d-leucine (1), a polyketide amino acid conjugate that inhibits Arabidopsis root growth. The biosynthesis of 1 includes a PKS-NRPS enzyme to assemble an N-acyl amino alcohol intermediate, which is further oxidized to an N-acyl amino acid (NAAA), demonstrating a new biosynthetic logic for synthesizing NAAAs and expanding the chemical space of products encoded by fungal PKS-NRPS clusters.

Identification and characterisation of a novel amino acid conjugate; ergothioneine conjugate of raloxifene through in vitro and in vivo studies

Identification and characterisation of a novel amino acid conjugate; ergothioneine conjugate of raloxifene through in vitro and in vivo studies

Neuroactive metabolites and bile acids are altered in extremely premature infants with brain injury

The gut microbiome is associated with pathological neurophysiological evolvement in extremely premature infants suffering from brain injury. The exact underlying mechanism and its associated metabolic signatures in infants are not fully understood. To decipher metabolite profiles linked to neonatal brain injury, we investigate the fecal and plasma metabolome of samples obtained from a cohort of 51 extremely premature infants at several time points, using liquid chromatography (LC)-high-resolution mass spectrometry (MS)-based untargeted metabolomics and LC-MS/MS-based targeted analysis for investigating bile acids and amidated bile acid conjugates. The data are integrated with 16S rRNA gene amplicon gut microbiome profiles as well as patient cytokine, growth factor, and Tcell profiles. We find an early onset of differentiation in neuroactive metabolites between infants with and without brain injury. We detect several bacterially derived bile acid amino acid conjugates in plasma and feces. These results provide insights into the early-life metabolome of extremely premature infants.

Bile salt hydrolase acyltransferase activity expands bile acid diversity.

Bile acids (BAs) are steroid detergents in bile that contribute to the absorption of fats and fat-soluble vitamins while shaping the gut microbiome because of their antimicrobial properties1-4. Here we identify the enzyme responsible for a mechanism of BA metabolism by the gut microbiota involving amino acid conjugation to the acyl-site of BAs, thus producing a diverse suite of microbially conjugated bile acids (MCBAs). We show that this transformation is mediated by acyltransferase activity of bile salt hydrolase (bile salt hydrolase/transferase,BSH/T). Clostridium perfringens BSH/T rapidly performed acyl transfer when provided various amino acids and taurocholate, glycocholate or cholate, with an optimum at pH 5.3. Amino acid conjugation by C. perfringens BSH/T was diverse, including all proteinaceous amino acids except proline and aspartate. MCBA production was widespread among gut bacteria, with strain-specific amino acid use. Species with similar BSH/T amino acid sequences had similar conjugation profiles and several bsh/t alleles correlated with increased conjugation diversity. Tertiary structure mapping of BSH/T followed by mutagenesis experiments showed that active site structure affects amino acid selectivity. These MCBA products had antimicrobial properties, where greater amino acid hydrophobicity showed greater antimicrobial activity. Inhibitory concentrations of MCBAs reached those measured natively in the mammalian gut. MCBAs fed to mice entered enterohepatic circulation, in which liver and gallbladder concentrations varied depending on the conjugated amino acid. Quantifying MCBAs in human faecal samples showed that they reach concentrations equal to or greater than secondary and primary BAs and were reduced after bariatric surgery, thus supporting MCBAs as a significant component of the BA pool that can be altered by changes in gastrointestinal physiology. In conclusion, the inherent acyltransferase activity of BSH/T greatly diversifies BA chemistry, creating a set of previously underappreciated metabolites with the potential to affect the microbiome and human health.

Identification of novel inhibitors of plant GH3 IAA-amido synthetases through molecular docking studies.

Auxins play a critical role in several plant developmental processes and their endogenous levels are regulated at multiple levels. The enzymes of the GRETCHEN HAGEN 3 (GH3) protein family catalyze the conjugation of amino acids to indoleacetic acid (IAA), the major endogenous auxin. The GH3 proteins are encoded by multiple redundant genes in plant genomes, making it difficult to perform functional genetic studies to understand their role in auxin homeostasis. To address these challenges, we used a chemical approach that exploits the reaction mechanism of GH3 proteins to identify small molecule inhibitors of their activity from a defined chemical library. The study evaluated receptor-ligand complexes based on their binding energy and classified them accordingly. Docking algorithms were used to correct any deviations, resulting in a list of the most important inhibitory compounds for selected GH3 enzymes based on a normalized sum of energy. The study presents atomic details of protein-ligand interactions and quantifies the effect of several of the identified small molecule inhibitors on auxin-mediated root growth processes in Arabidopsis thaliana. The direct effect of these compounds on endogenous auxin levels was measured using appropriate auxin sensors and endogenous hormone measurements. Our study has identified novel compounds of the flavonoid biosynthetic pathway that are effective inhibitors of GH3 enzyme-mediated IAA conjugation. These compounds play a versatile role in hormone-regulated plant development and have potential applications in both basic research and agriculture.