Structural homology of mite profilins to plant profilins is not indicative of allergic cross-reactivity.

Structural and allergenic characterization of mite profilins has not been previously pursued to a similar extent as plant profilins. Here, we describe structures of profilins originating from Tyrophagus putrescentiae (registered allergen Tyr p 36.0101) and Dermatophagoides pteronyssinus (here termed Der p profilin), which are the first structures of profilins from Arachnida. Additionally, the thermal stabilities of mite and plant profilins are compared, suggesting that the high number of cysteine residues in mite profilins may play a role in their increased stability. We also examine the cross-reactivity of plant and mite profilins as well as investigate the relevance of these profilins in mite inhalant allergy. Despite their high structural similarity to other profilins, mite profilins have low sequence identity with plant and human profilins. Subsequently, these mite profilins most likely do not display cross-reactivity with plant profilins. At the same time the profilins have highly conserved poly(l-proline) and actin binding sites.

Microscopic Menaces: The Impact of Mites on Human Health.

Mites are highly prevalent arthropods that infest diverse ecological niches globally. Approximately 55,000 species of mites have been identified but many more are yet to be discovered. Of the ones we do know about, most go unnoticed by humans and animals. However, there are several species from the Acariformes superorder that exert a significant impact on global human health. House dust mites are a major source of inhaled allergens, affecting 10-20% of the world's population; storage mites also cause a significant allergy in susceptible individuals; chiggers are the sole vectors for the bacterium that causes scrub typhus; Demodex mites are part of the normal microfauna of humans and their pets, but under certain conditions populations grow out of control and affect the integrity of the integumentary system; and scabies mites cause one of the most common dermatological diseases worldwide. On the other hand, recent genome sequences of mites provide novel tools for mite control and the development of new biomaterial with applications in biomedicine. Despite the palpable disease burden, mites remain understudied in parasitological research. By better understanding mite biology and disease processes, researchers can identify new ways to diagnose, manage, and prevent common mite-induced afflictions. This knowledge can lead to improved clinical outcomes and reduced disease burden from these remarkably widespread yet understudied creatures.

Precision engineering for localization, validation, and modification of allergenic epitopes.

The allergen-IgE interaction is essential for the genesis of allergic responses, yet investigation of the molecular basis of these interactions is in its infancy. Precision engineering has unveiled the molecular features of allergen-antibody interactions at the atomic level. High-resolution technologies, including x-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy, determine allergen-antibody structures. X-ray crystallography of an allergen-antibody complex localizes in detail amino acid residues and interactions that define the epitope-paratope interface. Multiple structures involving murine IgG mAbs have recently been resolved. The number of amino acids forming the epitope broadly correlates with the epitope area. The production of human IgE mAbs from B cells of allergic subjects is an exciting recent development that has for the first time enabled an actual IgE epitope to be defined. The biologic activity of defined IgE epitopes can be validated in vivo in animal models or by measuring mediator release from engineered basophilic cell lines. Finally, gene-editing approaches using the Clustered Regularly Interspaced Short Palindromic Repeats technology to either remove allergen genes or make targeted epitope engineering at the source are on the horizon. This review presents an overview of the identification and validation of allergenic epitopes by precision engineering.

At the outer part of the active site in Trypanosoma cruzi glucokinase: The role of phenylalanine 337.

The hole mutagenesis approach was used to interrogate the importance of F337 in Trypanosoma cruzi glucokinase (TcGlcK) in order to understand the complete set of binding interactions that are made by d-glucosamine analogue inhibitors containing aromatic tail groups that can extend to the outer part of the active site. An interesting inhibitor of this analogue class includes 2-N-carboxybenzyl-2-deoxy-d-glucosamine (CBZ-GlcN), which exhibits strong TcGlcK binding with a Ki of 710 nM. The residue F337 is found at the outer part of the active site that stems from the second protein subunit of the homodimeric assembly. In this study, F337 was changed to leucine and alanine so as to diminish phenylalanine's side chain size and attenuate intermolecular interactions in this region of the binding cavity. Results from enzyme - inhibitor assays revealed that the phenyl group of F337 made dominant hydrophobic interactions with the phenyl group of CBZ-GlcN as opposed to π - π stacking interactions. Moreover, enzymatic activity assays and X-ray crystallographic experiments indicated that each of these site-directed mutants primarily retained their activity and had high structural similarity of their protein fold. A computed structure model of T. cruzi hexokinase (TcHxK), which was produced by the artificial intelligence system AlphaFold, was compared to an X-ray crystal structure of TcGlcK. Our structural analysis revealed that TcHxK lacked an F337 counterpart residue and probably exists in the monomeric form. We proposed that the d-glucosamine analogue inhibitors that are structurally similar to CBZ-GlcN may not bind as strongly in TcHxK as they do in TcGlcK because of absent van der Waals contact from residue side chains.

Structural and functional studies reveal the molecular basis of substrate promiscuity of a glycosyltransferase originating from a major agricultural pest.

The two-spotted spider mite, Tetranychus urticae, is a major cosmopolitan pest that feeds on more than 1100 plant species. Its genome contains an unprecedentedly large number of genes involved in detoxifying and transporting xenobiotics, including 80 genes that code for UDP glycosyltransferases (UGTs). These enzymes were acquired via horizontal gene transfer from bacteria after loss in the Chelicerata lineage. UGTs are well-known for their role in phase II metabolism; however, their contribution to host adaptation and acaricide resistance in arthropods, such as T. urticae, is not yet resolved. TuUGT202A2 (Tetur22g00270) has been linked to the ability of this pest to adapt to tomato plants. Moreover, it was shown that this enzyme can glycosylate a wide range of flavonoids. To understand this relationship at the molecular level, structural, functional, and computational studies were performed. Structural studies provided specific snapshots of the enzyme in different catalytically relevant stages. The crystal structure of TuUGT202A2 in complex with UDP-glucose was obtained and site-directed mutagenesis paired with molecular dynamic simulations revealed a novel lid-like mechanism involved in the binding of the activated sugar donor. Two additional TuUGT202A2 crystal complexes, UDP-(S)-naringenin and UDP-naringin, demonstrated that this enzyme has a highly plastic and open-ended acceptor-binding site. Overall, this work reveals the molecular basis of substrate promiscuity of TuUGT202A2 and provides novel insights into the structural mechanism of UGTs catalysis.

Experimental and Computational Studies Reveal Novel Interaction of Lymphocytes Antigen 6K to TGF-ß Receptor Complex.

TGF-ß signaling promotes migration, invasion, and distant colonization of cancer cells in advanced metastatic cancers. TGF-ß signaling suppresses the anti-tumor immune response in a tumor microenvironment, allowing sustained tumor growth. TGF-ß plays an important role in normal physiology; thus it is no surprise that the clinical development of effective and safe TGF-ß inhibitors has been hampered due to their high toxicity. We discovered that increased expression of LY6K in cancer cells led to increased TGF-ß signaling and that inhibition of LY6K could lead to reduced TGF-ß signaling and reduced in vivo tumor growth. LY6K is a highly cancer-specific protein, and it is not expressed in normal organs except in the testes. Thus, LY6K is a valid target for developing therapeutic strategies to inhibit TGF-ß signaling in cancer cells. We employed in vitro pull-down assays and molecular dynamics simulations to understand the structural determinants of the TGF-ß receptor complex with LY6K. This combined approach allowed us to identify the critical residues and dynamics of the LY6K interaction with the TGF-ß receptor complex. These data are critical in designing novel drugs for the inhibition of TGF-ß in LY6K expressing cancer, induction of anti-tumor immune response, and inhibition of tumor growth and metastatic spread.

Membrane-Active Metallopolymers: Repurposing and Rehabilitating Antibiotics to Gram-Negative Superbugs.

Among multiple approaches to combating antimicrobial resistance, a combination therapy of existing antibiotics with bacterial membrane-perturbing agents is promising. A viable platform of metallopolymers as adjuvants in combination with traditional antibiotics is reported in this work to combat both planktonic and stationary cells of Gram-negative superbugs and their biofilms. Antibacterial efficacy, toxicity, antibiofilm activity, bacterial resistance propensity, and mechanisms of action of metallopolymer-antibiotic combinations are investigated. These metallopolymers exhibit 4-16-fold potentiation of antibiotics against Gram-negative bacteria with negligible toxicity toward mammalian cells. More importantly, the lead combinations (polymer-ceftazidime and polymer-rifampicin) eradicate preformed biofilms of MDR E. coli and P. aeruginosa, respectively. Further, ß-lactamase inhibition, outer membrane permeabilization, and membrane depolarization demonstrate synergy of these adjuvants with different antibiotics. Moreover, the membrane-active metallopolymers enable the antibiotics to circumvent bacterial resistance development. Altogether, the results indicate that such non-antibiotic adjuvants bear the promise to revitalize the efficacy of existing antibiotics to tackle Gram-negative bacterial infections.

Role of Small Molecule Ligands in IgE-Mediated Allergy.

PURPOSE OF REVIEW: A significant fraction of allergens bind small molecular ligands, and many of these compounds are classified as lipids. However, in most cases, we do not know the role that is played by the ligands in the allergic sensitization or allergic effector phases. RECENT FINDINGS: More effort is dedicated toward identification of allergens' ligands. This resulted in identification of some lipidic compounds that can play active immunomodulatory roles or impact allergens' molecular and allergic properties. Four allergen families (lipocalins, NPC2, nsLTP, and PR-10) are among the best characterized in terms of their ligand-binding properties. Allergens from these four families are able to bind many chemically diverse molecules. These molecules can directly interact with human immune system and/or affect conformation and stability of allergens. While there is more data on the allergens and their small molecular ligands, we are just starting to understand their role in allergy.

Molecular Basis of Plant Profilins' Cross-Reactivity.

Profilins are ubiquitous allergens with conserved structural elements. Exposure to profilins from different sources leads to IgE-cross-reactivity and the pollen-latex-food syndrome. Monoclonal antibodies (mAbs) that cross-react with plant profilins and block IgE-profilin interactions are relevant for diagnosis, epitope mapping, and specific immunotherapy. We generated IgGs mAbs, 1B4, and 2D10, against latex profilin (anti-rHev b 8) that inhibit the interaction of IgE and IgG4 antibodies from sera of latex- and maize-allergic patients by 90% and 40%, respectively. In this study, we evaluated 1B4 and 2D10 recognition towards different plant profilins, and mAbs recognition of rZea m 12 mutants by ELISAs. Interestingly, 2D10 highly recognized rArt v 4.0101 and rAmb a 8.0101, and to a lesser extent rBet v 2.0101, and rFra e 2.2, while 1B4 showed recognition for rPhl p 12.0101 and rAmb a 8.0101. We demonstrated that residue D130 at the α-helix 3 in profilins, which is part of the Hev b 8 IgE epitope, is essential for the 2D10 recognition. The structural analysis suggests that the profilins containing E130 (rPhl p 12.0101, rFra e 2.2, and rZea m 12.0105) show less binding with 2D10. The distribution of negative charges on the profilins' surfaces at the α-helices 1 and 3 is relevant for the 2D10 recognition, and that may be relevant to explain profilins' IgE cross-reactivity.

EAACI Molecular Allergology User's Guide 2.0.

Since the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE-mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the "EAACI Molecular Allergology User's Guide" (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure.

Lymphocyte antigen 6K signaling to aurora kinase promotes advancement of the cell cycle and the growth of cancer cells, which is inhibited by LY6K-NSC243928 interaction.

Lymphocyte antigen 6K (LY6K) is a small GPI-linked protein that is normally expressed in testes. Increased expression of LY6K is significantly associated with poor survival outcomes in many solid cancers, including cancers of the breast, ovary, gastrointestinal tract, head and neck, brain, bladder, and lung. LY6K is required for ERK-AKT and TGF-ß pathways in cancer cells and is required for in vivo tumor growth. In this report, we describe a novel role for LY6K in mitosis and cytokinesis through aurora B kinase and its substrate histone H3 signaling axis. Further, we describe the structural basis of the molecular interaction of small molecule NSC243928 with LY6K protein and the disruption of LY6K-aurora B signaling in cell cycle progression due to LY6K-NSC243928 interaction. Overall, disruption of LY6K function via NSC243928 led to failed cytokinesis, multinucleated cells, DNA damage, senescence, and apoptosis of cancer cells. LY6K is not required for vital organ function, thus inhibition of LY6K signaling is an ideal therapeutic approach for hard-to-treat cancers that lack targeted therapy such as triple-negative breast cancer.

Development of fluorophore labeled or biotinylated anticancer small molecule NSC243928.

Small molecule NSC243928 binds with LY6K, a potential target for the treatment of triple-negative breast cancer, and induces cancer cell death with an unclear mechanism. We have developed chemical tools to identify the molecular mechanisms of NSC243928-LY6K interaction. Herein, we report on the development and synthesis of biotinylated and fluorophore-tethered derivatives of NSC243928 guided by docking studies and molecular dynamics. Surface plasmon resonance assay indicates that these derivatives retained a direct binding with LY6K protein. Confocal analysis revealed that nitrobenzoxadiazole (NBD) fluorophore tagged NSC243928 is retained in LY6K expressing cancer cells. These novel modified compounds will be employed in future in vitro and in vivo studies to understand the molecular mechanisms of NSC243928 mediated cancer cell death. These studies will pave the path for developing novel targeted therapeutics and understanding any potential side-effects of these treatments for hard-to-treat cancers such as triple-negative breast cancer or other cancers with high expression of LY6K.

Human Monoclonal IgE Antibodies-a Major Milestone in Allergy.

PURPOSE OF REVIEW: Bound to its high affinity receptor on mast cells and basophils, the IgE antibody molecule plays an integral role in the allergic reaction. Through interactions with the allergen, it provides the sensitivity and specificity parameters for cell activation and mediator release that produce allergic symptoms. Advancements in human hybridoma technologies allow for the generation and molecular definition of naturally occurring allergen-specific human IgE monoclonal antibodies. RECENT FINDINGS: A high-resolution structure of dust mite allergen Der p 2 in complex with Fab of the human IgE mAb 2F10 was recently determined using X-ray crystallography. The structure reveals the fine molecular details of IgE 2F10 binding its 750 Å2 conformational epitope on Der p 2. This review provides an overview of this major milestone in allergy, the first atomic resolution structure of an authentic human IgE epitope. The molecular insights that IgE epitopes provide will allow for structure-based design approaches to the development of novel diagnostics, antibody therapeutics, and immunotherapies.

Current status of structural studies of proteins originating from Arachnida.

Arthropods from class Arachnida constitute a large and diverse group with over 100,000 described species, and they are sources of many proteins that have a direct impact on human health. Despite the importance of Arachnida, few proteins originating from these organisms have been characterized in terms of their structure. Here we present a detailed analysis of Arachnida proteins that have their experimental structures determined and deposited to the Protein Data Bank (PDB). Our results indicate that proteins represented in the PDB are derived from a small number of Arachnida families, and two-thirds of Arachnida proteins with experimental structures determined are derived from organisms belonging to Buthidae, Ixodidae, and Theraphosidae families. Moreover, 90% of the deposits come from just a dozen of Arachnida families, and almost half of the deposits represent proteins originating from only fifteen different species. In summary, our analysis shows that the structural analysis of proteins originating from Arachnida is not only limited to a small number of the source species, but also proteins from this group of animals are not extensively studied. However, the interest in Arachnida proteins seems to be increasing, which is reflected by a significant increase in the related PDB deposits during the last ten years.

Evaluation of the theoretical risk of cross-reactivity among recently identified food allergens for dogs.

BACKGROUND: There is increasing evidence of cross-reactivity between allergens of close or distant species. The A-RISC (Allergens'-Relative Identity, Similarity and Cross-reactivity) index helps evaluate the risk of theoretical cross-reactivity between proteins of the same family among different species. OBJECTIVES: To report the A-RISC indices for several food allergens of dogs between multiple food sources. MATERIALS AND METHODS: We selected several recently characterised food allergens for dogs from fish and chicken (ACTA1, ALDOA, CKM, ENO3, GAPDH, PKM and TPI1), fish (TPM1/2), beef/lamb (PGM1) and corn/potato (WAXY). When quality sequence data were available, A-RISC indices were calculated between multiple animal and plant species that can be used as food sources. For the TPM subunits, A-RISC indices also were calculated with the environmental allergens Bla g 4 and Der f 10, and the Toxocara canis nematode. RESULTS: The A-RISC indices suggest a substantial theoretical risk of cross-reactivity between species for all allergens considered. For TPM, this risk also extends to the environmental and nematode allergens. CONCLUSIONS AND CLINICAL RELEVANCE: There is a high theoretical risk of cross-reactivity between allergens of different species used as food sources. The clinical relevance of these elevated A-RISC indices should be studied further.

Human IgE monoclonal antibody recognition of mite allergen Der p 2 defines structural basis of an epitope for IgE cross-linking and anaphylaxis in vivo.

Immunoglobulin E (IgE) antibody is a critical effector molecule for adaptive allergen-induced immune responses, which affect up to 40% of the population worldwide. Allergens are usually innocuous molecules but induce IgE antibody production in allergic subjects. Allergen cross-linking of IgE bound to its high affinity receptor (FcεRI) on mast cells and basophils triggers release of histamine and other mediators that cause allergic symptoms. Little is known about the direct allergen-IgE antibody interaction due to the polyclonal nature of serum IgE and the low frequency of IgE-producing B cells in blood. Here, we report the X-ray crystal structure of a house dust mite allergen, Der p 2, in complex with Fab of a human IgE monoclonal antibody (mAb) isolated by hybridoma technology using human B cells from an allergic subject. This IgE mAb, 2F10, has the correct pairing of heavy and light chains as it occurs in vivo. Key amino acids forming the IgE epitope on Der p 2 were identified. Mutation of these residues ablated their functional ability to cross-link IgE in a mouse model of passive systemic anaphylaxis. These analyses revealed an important conformational epitope associated with the IgE antibody repertoire to a major mite allergen.

Intradiol ring cleavage dioxygenases from herbivorous spider mites as a new detoxification enzyme family in animals.

BACKGROUND: Generalist herbivores such as the two-spotted spider mite Tetranychus urticae thrive on a wide variety of plants and can rapidly adapt to novel hosts. What traits enable polyphagous herbivores to cope with the diversity of secondary metabolites in their variable plant diet is unclear. Genome sequencing of T. urticae revealed the presence of 17 genes that code for secreted proteins with strong homology to "intradiol ring cleavage dioxygenases (DOGs)" from bacteria and fungi, and phylogenetic analyses show that they have been acquired by horizontal gene transfer from fungi. In bacteria and fungi, DOGs have been well characterized and cleave aromatic rings in catecholic compounds between adjacent hydroxyl groups. Such compounds are found in high amounts in solanaceous plants like tomato, where they protect against herbivory. To better understand the role of this gene family in spider mites, we used a multi-disciplinary approach to functionally characterize the various T. urticae DOG genes. RESULTS: We confirmed that DOG genes were present in the T. urticae genome and performed a phylogenetic reconstruction using transcriptomic and genomic data to advance our understanding of the evolutionary history of spider mite DOG genes. We found that DOG expression differed between mites from different plant hosts and was induced in response to jasmonic acid defense signaling. In consonance with a presumed role in detoxification, expression was localized in the mite's gut region. Silencing selected DOGs expression by dsRNA injection reduced the mites' survival rate on tomato, further supporting a role in mitigating the plant defense response. Recombinant purified DOGs displayed a broad substrate promiscuity, cleaving a surprisingly wide array of aromatic plant metabolites, greatly exceeding the metabolic capacity of previously characterized microbial DOGs. CONCLUSION: Our findings suggest that the laterally acquired spider mite DOGs function as detoxification enzymes in the gut, disarming plant metabolites before they reach toxic levels. We provide experimental evidence to support the hypothesis that this proliferated gene family in T. urticae is causally linked to its ability to feed on an extremely wide range of host plants.

The first reptilian allergen and major allergen for fish-allergic patients: Crocodile ß-parvalbumin.

BACKGROUND: Clinical cross-reactivity between bony fish, cartilaginous fish, frog, and chicken muscle has previously been demonstrated in fish-allergic patients. In indicative studies, two reports of anaphylaxis following the consumption of crocodile meat and IgE-cross-binding were linked to the major fish allergen parvalbumin (PV). This study investigates IgE-binding proteins in crocodile meat with a focus on PV and their clinical relevance. METHODS: Proteins were extracted from muscle tissue of crocodile, three bony fish, and two cartilaginous fish. A cohort of fish-allergic pediatric patients (n = 77) underwent allergen skin prick testing (SPT) to three fish preparations (n = 77) and crocodile (n = 12). IgE-binding proteins were identified and quantified by SDS-PAGE, mass spectrometric analyses, and immunoblotting using commercial and in-house antibodies, as well as individual and pooled patients' serum. PV isoforms were purified or recombinantly expressed before immunological analyses, including human mast cell degranulation assay. RESULTS: Of the tissues analyzed, PV was most abundant in heated crocodile preparation, triggering an SPT of ≥3 mm in 8 of 12 (67%) fish-allergic patients. Seventy percent (31 of 44) of fish PV-sensitized patients demonstrated IgE-binding to crocodile PV. Crocodile ß-PV was the major IgE-binding protein but 20-fold less abundant than α-PV. Cellular reactivity was demonstrated for ß-PV and epitopes predicted, explaining frequent IgE-cross-binding of ß-PVs. Both PV isoforms are now registered as the first reptile allergens with the WHO/IUIS (ß-PV as Cro p 1 and α-PV as Cro p 2). CONCLUSION: Fish-allergic individuals may be at risk of an allergy to crocodile and should seek specialist advice before consuming crocodilian meat.

ß-Cyanoalanine synthase protects mites against Arabidopsis defenses.

Glucosinolates are antiherbivory chemical defense compounds in Arabidopsis (Arabidopsis thaliana). Specialist herbivores that feed on brassicaceous plants have evolved various mechanisms aimed at preventing the formation of toxic isothiocyanates. In contrast, generalist herbivores typically detoxify isothiocyanates through glutathione conjugation upon exposure. Here, we examined the response of an extreme generalist herbivore, the two-spotted spider mite Tetranychus urticae (Koch), to indole glucosinolates. Tetranychus urticae is a composite generalist whose individual populations have a restricted host range but have an ability to rapidly adapt to initially unfavorable plant hosts. Through comparative transcriptomic analysis of mite populations that have differential susceptibilities to Arabidopsis defenses, we identified ß-cyanoalanine synthase of T. urticae (TuCAS), which encodes an enzyme with dual cysteine and ß-cyanoalanine synthase activities. We combined Arabidopsis genetics, chemical complementation and mite reverse genetics to show that TuCAS is required for mite adaptation to Arabidopsis through its ß-cyanoalanine synthase activity. Consistent with the ß-cyanoalanine synthase role in detoxification of hydrogen cyanide (HCN), we discovered that upon mite herbivory, Arabidopsis plants release HCN. We further demonstrated that indole glucosinolates are sufficient for cyanide formation. Overall, our study uncovered Arabidopsis defenses that rely on indole glucosinolate-dependent cyanide for protection against mite herbivory. In response, Arabidopsis-adapted mites utilize the ß-cyanoalanine synthase activity of TuCAS to counter cyanide toxicity, highlighting the mite's ability to activate resistant traits that enable this extreme polyphagous herbivore to exploit cyanogenic host plants.

Structural and functional characterization of ß-cyanoalanine synthase from Tetranychus urticae.

Tetranychus urticae is a polyphagous spider mite that can feed on more than 1100 plant species including cyanogenic plants. The herbivore genome contains a horizontally acquired gene tetur10g01570 (TuCAS) that was previously shown to participate in cyanide detoxification. To understand the structure and determine the function of TuCAS in T. urticae, crystal structures of the protein with lysine conjugated pyridoxal phosphate (PLP) were determined. These structures reveal extensive TuCAS homology with the ß-substituted alanine synthase family, and they show that this enzyme utilizes a similar chemical mechanism involving a stable α-aminoacrylate intermediate in ß-cyanoalanine and cysteine synthesis. We demonstrate that TuCAS is more efficient in the synthesis of ß-cyanoalanine, which is a product of the detoxification reaction between cysteine and cyanide, than in the biosynthesis of cysteine. Also, the enzyme carries additional enzymatic activities that were not previously described. We show that TuCAS can detoxify cyanide using O-acetyl-L-serine as a substrate, leading to the direct formation of ß-cyanoalanine. Moreover, it catalyzes the reaction between the TuCAS-bound α-aminoacrylate intermediate and aromatic compounds with a thiol group. In addition, we have tested several compounds as TuCAS inhibitors. Overall, this study identifies additional functions for TuCAS and provides new molecular insight into the xenobiotic metabolism of T. urticae.