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Tyrosine aminotransferase is a well-characterized enzyme in the Leishmania parasite, but the role of TAT in the parasite functioning remains largely unknown. In this study, we attempt to gain a better understanding of the enzyme's role in the parasite by gene knockout and overexpression of the TAT gene. The overexpression of TAT protein was well tolerated by the parasites in two independent repeats. Single knockout of TAT gene by homologous recombination, LdTAT

Fumarylacetoacetate hydrolase (FAH) catalyzes the final step of Tyrosine (Tyr) degradation pathway essential to animals and the deficiency of FAH causes an inborn lethal disease. In plants, a role of this pathway was unknown until we found that mutation of Short-day Sensitive Cell Death1 (SSCD1), encoding Arabidopsis FAH, results in cell death under short day. Phenylalanine (Phe) could be converted to Tyr and then degraded in both animals and plants. Phe ingestion in animals worsens the disease caused by FAH defect. However, in this study we found that Phe represses cell death caused by FAH defect in plants. Phe treatment promoted chlorophyll biosynthesis and suppressed the up‑regulation of reactive oxygen species marker genes in the sscd1 mutant. Furthermore, the repression of sscd1 cell death by Phe could be reduced by α-aminooxi-β-phenylpropionic acid but increased by methyl jasmonate, which inhibits or activates Phe ammonia-lyase catalyzing the first step of phenylpropanoid pathway, respectively. In addition, we found that jasmonate signaling up‑regulates Phe ammonia-lyase 1 and mediates the methyl jasmonate enhanced repression of sscd1 cell death by Phe. These results uncovered the relation between chlorophyll biosynthesis, phenylpropanoid pathway and jasmonate signaling in regulating the cell death resulting from loss of FAH in plants.

Tyrosine aminotransferase (TAT) catalyzes the transamination of amino acids in Leishmania sp.. TAT from Leishmania donovani has been found to be extremely stable at extreme temperatures and pH conditions. This study was conceived to map the functions of the non-conserved N-terminal and conserved C-terminal domain of TAT. N-terminal (NTAT) and C-terminal (CTAT) domain of TAT was truncated and cloned into the pET28a(+) vector. The truncated proteins were expressed, purified, and biochemically characterized. The Km of NTAT and CTAT for the tyrosine-pyruvate pair was determined to be 3.468 ± 0.796 mM and 4.581 ± 0.627 mM, repectively. Temperature and pH stability studies found NTAT to be stable like TAT but CTAT was extremely susceptible to temperature and pH changes. Upon docking and simulation for 100 ns, NTAT had lower SASA values. From UV spectroscopic study, PLP bound better to CTAT than NTAT because of the reduced SASA of NTAT. The sensitivity of CTAT was reasoned when the urea denaturation studies showed two-state denaturation which differed from NTAT’s and TAT’s biphasic folding mechanism. From this study, the authors hypothesize that the N-terminal is responsible for PLP stabilization and C-terminal protects the active site from extreme conditions.

Main conclusion Tyrosine aminotransferase (AaTAT) from the hornwort Anthoceros agrestis Paton (Anthocerotaceae) was amplified and expressed in E. coli. The active enzyme is able to accept a wide range of substrates with distinct preference for l-tyrosine, therefore, possibly catalysing the initial step in rosmarinic acid biosynthesis. Abstract The presence of rosmarinic acid (RA) in the hornwort A. agrestis is well known, and some attempts have been made to clarify the biosynthesis of this caffeic acid ester in lower plants. Parallel to the biosynthesis in vascular plants, the involvement of tyrosine aminotransferase (EC 2.6.1.5; TAT) as the initial step was assumed. The amplification of a nucleotide sequence putatively encoding AaTAT (Genbank MN922307) and expression in E. coli were successful. The enzyme proved to have a high acceptance of l-tyrosine (Km 0.53 mM) whilst slightly preferring 2-oxoglutarate over phenylpyruvate as co-substrate. Applying l-phenylalanine as a potential amino donor or using oxaloacetate or pyruvate as a replacement for 2-oxoglutarate as amino acceptor resulted in significantly lower catalytic efficiencies in each of these cases. To facilitate further substrate search, two methods were introduced, one using ninhydrin after thin-layer chromatography and the other using derivatisation with o-phthalaldehyde followed by HPLC or LC–MS analysis. Both methods proved to be well applicable and helped to confirm the acceptance of further aromatic and aliphatic amino acids. This work presents the first description of a heterologously expressed TAT from a hornwort (A. agrestis) and describes the possible entry into the biosynthesis of RA and other specialised compounds in a so far neglected representative of terrestrial plants and upcoming new model organism.

Plants produce various l-tyrosine (Tyr)-derived compounds that are critical for plant adaptation and have pharmaceutical or nutritional importance for human health. Tyrosine aminotransferases (TATs) catalyze the reversible reaction between Tyr and 4-hydroxyphenylpyruvate (HPP), representing the entry point in plants for both biosynthesis of various natural products and Tyr degradation in the recycling of energy and nutrients. To better understand the roles of TATs and how Tyr is metabolized in planta, here we characterized single and double loss-of-function mutants of TAT1 (At5g53970) and TAT2 (At5g36160) in the model plant Arabidopsis thaliana. As reported previously, tat1 mutants exhibited elevated and decreased levels of Tyr and tocopherols, respectively. The tat2 mutation alone had no impact on Tyr and tocopherol levels, but a tat1 tat2 double mutant had increased Tyr accumulation and decreased tocopherol levels under high-light stress compared with the tat1 mutant. Relative to WT and the tat2 mutant, the tat1 mutant displayed increased vulnerability to continuous dark treatment, associated with an early drop in respiratory activity and sucrose depletion. During isotope-labeled Tyr feeding in the dark, we observed that the tat1 mutant exhibits much slower (13)C incorporation into tocopherols, fumarate, and other tricarboxylic acid (TCA) cycle intermediates than WT and the tat2 mutant. These results indicate that TAT1 and TAT2 function together in tocopherol biosynthesis, with TAT2 having a lesser role, and that TAT1 plays the major role in Tyr degradation in planta. Our study also highlights the importance of Tyr degradation under carbon starvation conditions during dark-induced senescence in plants.

Tyrosine aminotransferase (TAT, EC 2.6.1.5) is the first key enzyme that catalyzes the reversible interconversion of tyrosine and 4-hydroxyphenylpyruvate in the tyrosine-derived pathway for syntheses of important secondary metabolites and compounds. Although plant TAT genes have been proposed to be important in response to abiotic stress, there is little information about TAT genes in woody perennial tree species, especially in economic fruit trees. Based on TAT domain searching, sequence homology screening and phylogenetic analysis, we identified four TATs in apple genome. Then, we carried out a detailed phylogenetic analysis of TAT genes from multi-species, focusing on apple (Malus domestica). The result showed that the TAT family comprises three major classes corresponding to genes from angiosperms, mammals, and bacteria. Angiosperm TAT genes could be further divided into six subclasses. Analysis of intron-exon structure revealed that the typical TAT gene contains six introns and seven exons, with exons of similar size at each exon location. Promoter analysis showed that the 5'-flanking region of apple MdTATs contain multiple cis-acting elements including those implicated in light, biotic stress, abiotic stress, and hormone response. MdTATs were expressed to various levels in all apple structures and organs evaluated, and showed distinct expression patterns under water deficit stress. Ectopic expression of MdTAT2 in Arabidopsis or over-expression of MdTAT2 in apple callus tissue conferred enhanced tolerance to drought and osmotic stress. Collectively, these results suggest a role for TAT genes in drought and osmotic stresses and provide valuable information for further research of TAT genes and their function in plants.

Rosmarinic acid (RA) is an active constituent of

Rosmarinic acid is a hydroxycinnamic acid ester commonly found in the Boraginaceae and Lamiaceae plant families. It exhibits various biological activities, including antioxidant, anti-inflammatory, antibacterial, antiallergic, and antiviral properties. Rosmarinic acid is used as a food and cosmetic ingredient, and several pharmaceutical applications have been suggested as well. Rosmarinic acid is currently produced by extraction from plants or chemical synthesis; however, due to limited availability of the plant sources and the complexity of the chemical synthesis method, there is an increasing interest in producing this compound by microbial fermentation. In this study, we aimed to produce rosmarinic acid by engineered baker's yeast

The current drugs for treating Leishmaniasis are toxic, non-economical and with the emergence of drug resistance makes the need for novel therapeutics urgent and necessary. In the current study, we report the identification of compounds TI 1-5 against tyrosine aminotransferase of L. donovani from a curated ZINC15 database containing 183,659 compounds. These flavonoid compounds had binding energies-8 kcal/mol and interacted with the active site residues S151, K286, C290, and P291. Assessment of physicochemical descriptors and ADMET properties established the drug likeliness of these compounds. The all-atom molecular dynamic simulations of the TAT-TI complexes exhibited stable geometrical properties and further trajectory analysis revealed the high-affinity interactions of TI 1, 3, 4, and 5 with the active site residues. DFT calculations reported the high electrophilic nature of TI 2 while other TI compounds demonstrated good kinetic stability and reactivity. From in vitro studies, TI 3 and TI 4 had the highest inhibition with Ki values of 0.9 ± 0.2 μM and 0.30 ± 0.1 μM, respectively. Taken together, the results from this study indicate the potentiality of TI 1, 3, 4, and 5 as anti-leishmanial leads, and these compounds can be exploited to manage the growing Leishmaniasis crisis in the world.

Aging is characterized by extensive metabolic reprogramming. To identify metabolic pathways associated with aging, we analyzed age-dependent changes in the metabolomes of long-lived Drosophila melanogaster. Among the metabolites that changed, levels of tyrosine were increased with age in long-lived flies. We demonstrate that the levels of enzymes in the tyrosine degradation pathway increase with age in wild-type flies. Whole-body and neuronal-specific downregulation of enzymes in the tyrosine degradation pathway significantly extends Drosophila lifespan, causes alterations of metabolites associated with increased lifespan, and upregulates the levels of tyrosine-derived neuromediators. Moreover, feeding wild-type flies with tyrosine increased their lifespan. Mechanistically, we show that suppression of ETC complex I drives the upregulation of enzymes in the tyrosine degradation pathway, an effect that can be rescued by tigecycline, an FDA-approved drug that specifically suppresses mitochondrial translation. In addition, tyrosine supplementation partially rescued lifespan of flies with ETC complex I suppression. Altogether, our study highlights the tyrosine degradation pathway as a regulator of longevity.

The glucocorticoid status and activities of ALT, AST, and tyrosine aminotransferase in the liver are studied in rats with streptozotocin-induced diabetes mellitus in response to repeated intraperitoneal injections of mifepristone. Diabetic rats develop an increase of the blood corticosterone and liver aminotransferase levels in response to mifepristone. These results indicate that in diabetic animals the glucocorticoid hormones with high blood concentrations, increasing still more in response to mifepristone, overcome the receptor blockade, and realize their regulatory functions in hepatocytes. The effects of mifepristone on ALT activity are the most manifest. In normal rats, only ALT activity is increasing in response to mifepristone, while in rats with streptozotocin-induced diabetes mellitus, ALT activity increases more intensely than activities of tyrosine aminotransferase and AST.

Background In 2012, Venet et al. proposed that at least in the case of breast cancer, most published signatures are not significantly more associated with outcome than randomly generated signatures. They suggested that nominal p-value is not a good estimator to show the significance of a signature. Therefore, one can reasonably postulate that some information might be present in such significant random signatures. Methods In this research, first we show that, using an empirical p-value, these published signatures are more significant than their nominal p-values. In other words, the proposed empirical p-value can be considered as a complimentary criterion for nominal p-value to distinguish random signatures from significant ones. Secondly, we develop a novel computational method to extract information that are embedded within significant random signatures. In our method, a score is assigned to each gene based on the number of times it appears in significant random signatures. Then, these scores are diffused through a protein-protein interaction network and a permutation procedure is used to determine the genes with significant scores. The genes with significant scores are considered as the set of significant genes. Results First, we applied our method on the breast cancer dataset NKI to achieve a set of significant genes in breast cancer considering significant random signatures. Secondly, prognostic performance of the computed set of significant genes is evaluated using DMFS and RFS datasets. We have observed that the top ranked genes from this set can successfully separate patients with poor prognosis from those with good prognosis. Finally, we investigated the expression pattern of TAT, the first gene reported in our set, in malignant breast cancer vs. adjacent normal tissue and mammospheres. Conclusion Applying the method, we found a set of significant genes in breast cancer, including TAT, a gene that has never been reported as an important gene in breast cancer. Our results show that the expression of TAT is repressed in tumors suggesting that this gene could act as a tumor suppressor in breast cancer and could be used as a new biomarker.

A multiscale pharmacodynamic model was developed to characterize the receptor-mediated, transcriptomic, and proteomic determinants of corticosteroid (CS) effects on clinically relevant hepatic processes following a single dose of methylprednisolone (MPL) given to adrenalectomized (ADX) rats. The enhancement of tyrosine aminotransferase (TAT) mRNA, protein, and enzyme activity were simultaneously described. Mechanisms related to the effects of MPL on glucose homeostasis, including the regulation of CCAAT-enhancer binding protein-beta (C/EBPβ) and phosphoenolpyruvate carboxykinase (PEPCK) as well as insulin dynamics were evaluated. The MPL-induced suppression of circulating lymphocytes was modeled by coupling its effect on cell trafficking with pharmacogenomic effects on cell apoptosis via the hepatic (STAT3-regulated) acute phase response. Transcriptomic and proteomic time-course profiles measured in steroid-treated rat liver were utilized to model the dynamics of mechanistically relevant gene products, which were linked to associated systemic end-points. While time-courses of TAT mRNA, protein, and activity were well described by transcription-mediated changes, additional post-transcriptional processes were included to explain the lack of correlation between PEPCK mRNA and protein. The immune response model quantitatively discerned the relative roles of cell trafficking versus gene-mediated lymphocyte apoptosis by MPL. This systems pharmacodynamic model provides insights into the contributions of selected molecular events occurring in liver and explores mechanistic hypotheses for the multi-factorial control of clinically relevant pharmacodynamic outcomes.

A 65-year-old Caucasian woman presented to the emergency department with rapidly worsening shortness of breath. On presentation she was tachycardic and tachypnoeic with reduced (85%) oxygen saturation. Cardiovascular examination revealed elevated jugular venous pressure with positive Kussmaul's sign, pulsus paradoxus and muffled heart sounds. Her inflammatory markers were elevated; she had neutrophilia and deranged liver function tests. Imaging revealed cardiomegaly, a large fusiform thoracic aortic aneurysm, pericardial effusion and right ventricular free wall collapse during diastole (suggestive of tamponade). Urgent pericardiocentesis was performed with rapid symptomatic relief. She subsequently underwent aortic root and aortic valve replacement surgery. Histology of the resected specimen showed inflammatory infiltrate with giant cell formation indicative of giant cell arteritis (GCA). This case highlights the need to consider GCA in the differential diagnosis of patients presenting with aortic aneurysm and pericardial effusion.

The content of the amino acids in the placenta during physiological pregnancy and fetal growth restriction (FGR) has been investigated my means of the method of ion-exchange chromatography. It has been found that in FGR the placental amino acid pool is characterized by a decreased content of arginine, proline, alanine, serine, cysteine, methionine, tryptophan, leucine, threonine, tyrosine, phenylalanine, glutamine and an increased content of dicarboxylic amino acids, lysine, histidine and glycine. These changes are accompanied by altered activity of some enzymes of amino acid metabolism, and the degree of these changes correlates with the level of corresponding amino acids.S pomoshch'iu metoda ionoobmennoĭ khromatografii izucheno soderzhanie aminokislot v platsente pri fiziologicheskoĭ beremennosti i zaderzhke rosta ploda (ZRP). Ustanovleno, chto pri ZRP aminokislotnyĭ fond platsenty kharakterizuetsia snizheniem soderzhaniia arginina, prolina, alanina, serina, tsisteina, metionina, triptofana, leĭtsina, treonina, tirozina, fenilalanina i glutamina, uchastvuiushchikh vo mnogikh metabolicheskikh protsessakh, neobkhodimykh dlia podderzhaniia funktsionirovaniia fetoplatsentarnoĭ sistemy. Protivopolozhnye otkloneniia imeiut mesto dlia dikarbonovykh aminokislot, lizina, gistidina i glitsina, soderzhanie kotorykh povyshaetsia. Pri étom izmeniaetsia i aktivnost' riada fermentov aminokislotnogo obmena, prichem stepen' étikh izmeneniĭ korreliruet s urovnem sootvetstvuiushchikh aminokislot. Vazhnym sledstviem aminokislotnogo disbalansa v platsente pri ZRP iavliaetsia umen'shenie fiziologicheskogo sootnosheniia nezamenimykh i zamenimykh aminokislot, chto soprovozhdaetsia ukhudsheniem trofiki ploda i snizheniem protsessov ego rosta i razvitiia pri dannoĭ patologii gestatsii.

Plants produce various L-tyrosine (Tyr)-derived compounds that are of pharmaceutical or nutritional importance to humans. Tyr aminotransferase (TAT) catalyzes the reversible transamination between Tyr and 4-hydroxyphenylpyruvate (HPP), the initial step in the biosynthesis of many Tyr-derived plant natural products. Herein reported is the biochemical characterization and subcellular localization of TAT enzymes from the model plant Arabidopsis thaliana. Phylogenetic analysis showed that Arabidopsis has at least two homologous TAT genes, At5g53970 (AtTAT1) and At5g36160 (AtTAT2). Their recombinant enzymes showed distinct biochemical properties: AtTAT1 had the highest activity towards Tyr, while AtTAT2 exhibited a broad substrate specificity for both amino and keto acid substrates. Also, AtTAT1 favored the direction of Tyr deamination to HPP, whereas AtTAT2 preferred transamination of HPP to Tyr. Subcellular localization analysis using GFP-fusion proteins and confocal microscopy showed that AtTAT1, AtTAT2, and HPP dioxygenase (HPPD), which catalyzes the subsequent step of TAT, are localized in the cytosol, unlike plastid-localized Tyr and tocopherol biosynthetic enzymes. Furthermore, subcellular fractionation indicated that, while HPPD activity is restricted to the cytosol, TAT activity is detected in both cytosolic and plastidic fractions of Arabidopsis leaf tissue, suggesting that an unknown aminotransferase(s) having TAT activity is also present in the plastids. Biochemical and cellular analyses of Arabidopsis TATs provide a fundamental basis for future in vivo studies and metabolic engineering for enhanced production of Tyr-derived phytochemicals in plants.

Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine to p-hydroxyphenylpyruvate. Accumulation of tyrosine in the body due to a genetic mutation in the TAT gene causes tyrosomia type II in humans. The TAT gene is regarded as a model for studying steroid-inducible factors regulating a variety of biological functions of TAT. However, little is known of the effects of estrogen on the expression of the TAT gene in chickens. Therefore, in the present study, we identified expression of the avian TAT gene in various organs. The results showed the TAT was detected predominantly in the liver and reproductive organs including testis, oviduct, and ovary. Specifically, TAT mRNA was expressed abundantly in the glandular and luminal epithelia of the oviducts in response to endogenous and exogenous estrogens which also induce dramatic morphological changes in the oviduct of chickens. In addition, target microRNAs of TAT (miR-1460, miR-1626-3p, miR-1690-5p, and miR-7442-3p) were found to modulate expression of the TAT gene. Especially, miR-1690-5p influenced TAT gene transcription by binding directly to its 3'-UTR region. Moreover, the expression of TAT was abundant in glandular epithelia of cancerous but not normal ovaries from laying hens. Taken together, our findings suggest that TAT plays an important role in the cytodifferentiation of oviducts in response to estrogen and in the progression of ovarian cancer in chickens.

The current study was undertaken to investigate the spectrum of tyrosine transaminases enzymes in a cytosolic fraction of rat brain and to specifically purify and characterize a previously identified cytosolic brain enzyme possessing tyrosine/glyoxylate transaminase activity. Based upon extensive biochemical and immunochemical characterization of purified brain tyrosine/glyoxylate transaminase, we concluded the purified enzyme is glutamine transaminase-K (EC 2.6.1.64). This conclusion was based on: 1.) a concurrent enrichment in the tyrosine/glyoxylate and glutamine/phenylpyruvate transaminase activities during purification, 2.) demonstration of a co-substrate specificity for amino acids and α-keto acids that was highly consistent with published information for glutamine transaminase-K, 3.) results from detailed kinetic analysis, 4.) glutamine was a potent inhibitor of in vitro tyrosine/glyoxylate transamination, 5.) biochemical characterization, including pH optimum of 8.5 and spectrophotometric analysis and 6.) immunoanalytical analysis using a specific antiserum to rat renal glutamine transaminase-k. In addition, immunochemical characterization of a crude soluble extract of whole brain suggests that the in vitro tyrosine transaminase activity for several different α-keto acid co-substrates likely reflect the activity of glutamine transaminase-K. In conclusion, this investigation confirmed the presence of multiple tyrosine transaminase enzymes in a cytosolic extract of rat brain. Moreover, we concluded glutamine transaminase-K represents a predominant cytosolic enzyme in rat brain that's capable of catalyzing in vitro transamination of p-tyrosine and other aromatic amino acids, including the neurotransmitter precursors L-dopa and 5-hydroxytryptophan. The purified transaminase possesses a broad co-substrate specificity with preferential reactivity with α-keto acids derived from neutral aliphatic and aromatic amino acids. Lastly, we identified a heterogeneous regional distribution of tyrosine/glyoxylate transaminase (glutamine transaminase-K) in rat brain with a significantly higher level of in vitro activity in cerebellum.

OBJECTIVEDespite an aggressive multimodal therapeutic regimen, glioblastoma (GBM) continues to portend a grave prognosis, which is driven in part by tumor heterogeneity at both the molecular and cellular levels. Accordingly, herein the authors sought to identify metabolic differences between GBM tumor core cells and edge cells and, in so doing, elucidate novel actionable therapeutic targets centered on tumor metabolism.METHODSComprehensive metabolic analyses were performed on 20 high-grade glioma (HGG) tissues and 30 glioma-initiating cell (GIC) sphere culture models. The results of the metabolic analyses were combined with the Ivy GBM data set. Differences in tumor metabolism between GBM tumor tissue derived from within the contrast-enhancing region (i.e., tumor core) and that from the peritumoral brain lesions (i.e., tumor edge) were sought and explored. Such changes were ultimately confirmed at the protein level via immunohistochemistry.RESULTSMetabolic heterogeneity in both HGG tumor tissues and GBM sphere culture models was identified, and analyses suggested that tyrosine metabolism may serve as a possible therapeutic target in GBM, particularly in the tumor core. Furthermore, activation of the enzyme tyrosine aminotransferase (TAT) within the tyrosine metabolic pathway influenced the noted therapeutic resistance of the GBM core.CONCLUSIONSSelective inhibition of the tyrosine metabolism pathway may prove highly beneficial as an adjuvant to multimodal GBM therapies.

Stem cells expressing reporter constructs are extremely useful for their tracking in vivo or for determining cell lineage fate in vivo and in vitro. We generated liver progenitor cell (LPC) lines from actin-EGFP and TAT-GRE-lacZ mice. LPCs from the actin-EGFP mouse facilitate cell tracing following transplant as the reporter is constitutively expressed. LPCs from the TAT-GRE-lacZ mouse express β-galactosidase under the control of the tyrosine aminotransferase (TAT) promoter and are only active in mature hepatocytes. We found that the utility of such LPC lines becomes severely limited by downregulation of transgene expression following extended culture. We show that epigenetic mechanisms are responsible for suppressing expression of both transgenes. Enhancement of transgene expression in both LPC lines was achieved by treating the cell lines with either the histone acetylating agent sodium butyrate or the DNA demethylating agent 5-azacytidine.

Leishmania donovani tyrosine aminotransferase (LdTAT) is an essential enzyme that catalyzes the first step of amino acid catabolism. To understand LdTAT activity at different pH, molecular dynamics simulations were performed and trajectory and T-pad analysis pad were conducted. Fluorescence spectroscopy of LdTAT at various pH was measured to understand structural stability. UV studies on PLP were performed to determine the binding of the enzyme to cofactor PLP at different pH. The MD simulations showed that the structure of LdTAT was stable and no structural denaturation was observed at pH 2, 7 and 12. LdTAT exhibited the highest activity at pH -8 and fluorescent spectroscopy also corroborated by exhibiting the highest intensity at pH -8. Moreover, no structural denaturation was observed during the pH gradient. UV studies concluded that the aldimine bond forms only around neutral pH and redshift was observed on enzyme binding. From our observation, we hypothesize that the activity of LdTAT is a close interplay between the structure and charges of K286 and PLP. This study may provide significant insight into understanding parasitic enzymes like LdTAT during the life-cycle of Leishmania parasite. Knowledge of such enzyme mechanisms can pave the way for the design and delivery of enzyme-specific inhibitors.

One strain of endophytic fungus ZPRa-R-1 was obtained for the capacity of promoting production of salidroside in Rhodiola crenulata. To explain the mechanism of salidroside biosynthesis in host plant, eight housekeeping genes were evaluated, and the evaluation method was created for the expression activities of four key enzyme genes PAL (phenylalanine ammonia-lyase), TyDC (tyrosine decarboxylase), TAT (tyrosine transaminase), UDPGT (UDP-glucosyltransferase) referenced double reference genes in biosynthesis pathway of salidroside in R. crenulata. Stabilities of housekeeping genes were confirmed by real-time fluorescent quantitative PCR technology and three softwares including geNorm, NormFinder and BestKeeper, then relative expressions of key enzyme genes were analysized by the 2-ΔΔCt method. The results showed that the most stable gene was GAPDH, followed by PCS, and the most appropriate reference of internal genes were combination with two genes in R. crenulata inoculated with endophytic fungus ZPRa-R-1. Under symbiosis conditions, regularity changes of key enzyme genes affected by endophytic fungus ZPRa-R-1 were as follows: the relative expression activity of PAL attached to peak value, which was 4.9 times as that of control group when inoculated ten days. The relative expression of TyDC reached the maximum value, which was 2.8 times of that control after inoculating 12 days. The relative expression of UDPGT actually reach 17.1 times than that of control after inoculating 8 days. However, the relative expression of TAT was not affected by this fungus. The changes of four key enzyme genes are positively correlated with the changes of salidroside content in R. crenulata.

Under oxidative stress conditions, hydroxyl radicals can oxidize the phenyl ring of phenylalanine, producing the abnormal tyrosine isomer meta-tyrosine (m-tyrosine). m-Tyrosine levels are commonly used as a biomarker of oxidative stress, and its accumulation has recently been reported to adversely affect cells, suggesting a direct role for m-tyrosine in oxidative stress effects. We found that the Caenorhabditis elegans ortholog of tyrosine aminotransferase (TATN-1)—the first enzyme involved in the metabolic degradation of tyrosine—is up-regulated in response to oxidative stress and directly activated by the oxidative stress–responsive transcription factor SKN-1. Worms deficient in tyrosine aminotransferase activity displayed increased sensitivity to multiple sources of oxidative stress. Biochemical assays revealed that m-tyrosine is a substrate for TATN-1–mediated deamination, suggesting that TATN-1 also metabolizes m-tyrosine. Consistent with a toxic effect of m-tyrosine and a protective function of TATN-1, tatn-1 mutant worms exhibited delayed development, marked reduction in fertility, and shortened lifespan when exposed to m-tyrosine. A forward genetic screen identified a mutation in the previously uncharacterized gene F01D4.5—homologous with human transcription factor 20 (TCF20) and retinoic acid–induced 1 (RAI1)—that suppresses the adverse phenotypes observed in m-tyrosine–treated tatn-1 mutant worms. RNA-Seq analysis of F01D4.5 mutant worms disclosed a significant reduction in the expression of specific isoforms of genes encoding ribosomal proteins, suggesting that alterations in protein synthesis or ribosome structure could diminish the adverse effects of m-tyrosine. Our findings uncover a critical role for tyrosine aminotransferase in the oxidative stress response via m-tyrosine metabolism.

Tocopherols composed of four isoforms (α, β, γ, and δ) and its biosynthesis comprises of three pathways: methylerythritol 4-phosphate (MEP), shikimate (SK) and tocopherol-core pathways regulated by 25 enzymes. To understand pathway regulatory mechanism at transcriptional level, gene expression profile of tocopherol-biosynthesis genes in two soybean genotypes was carried out, the results showed significantly differential expression of 5 genes: 1-deoxy-d-xylulose-5-P-reductoisomerase (DXR), geranyl geranyl reductase (GGDR) from MEP, arogenate dehydrogenase (TyrA), tyrosine aminotransferase (TAT) from SK and γ-tocopherol methyl transferase 3 (γ-TMT3) from tocopherol-core pathways. Expression data were further analyzed for total tocopherol (T-toc) and α-tocopherol (α-toc) content by coregulation network and gene clustering approaches, the results showed least and strong association of γ-TMT3/tocopherol cyclase (TC) and DXR/DXS, respectively, with gene clusters of tocopherol biosynthesis suggested the specific role of γ-TMT3/TC in determining tocopherol accumulation and intricacy of DXR/DXS genes in coordinating precursor pathways toward tocopherol biosynthesis in soybean seeds. Thus, the present study provides insight into the major role of these genes regulating the tocopherol synthesis in soybean seeds.

Endophyte is a factor that affects the physiology and metabolism of plant. However, limited information is available on the mechanism of interaction between endophyte and plant. To investigate the effects of endophytic fungus ZPRs-R11, that is, Trimmatostroma sp., on salidroside and tyrosol accumulations in Rhodiola crenulata, signal transduction, enzyme gene expression, and metabolic pathway were investigated. Results showed that hydrogen peroxide (H2O2), nitric oxide (NO), and salicylic acid (SA) involved in fungus-induced salidroside and tyrosol accumulations. NO acted as an upstream signal of H2O2 and SA. No up- or down-stream relationship was observed, but mutual coordination existed between H2O2 and SA. Rate-limiting enzyme genes with the maximum expression activities were UDP-glucosyltransferase, tyrosine decarboxylase (TYDC), monoamine oxidase, phenylalanine ammonialyase (PAL), and cinnamic-4-hydroxylase sequentially. Nevertheless, the genes of tyrosine transaminase and pyruvate decarboxylase only indicated slightly higher activities than those in control. Thus, TYDC and PAL branches were the preferential pathways in ZPRs-R11-induced salidroside and tyrosol accumulation. Trimmatostroma sp. was a potential fungus for promoting salidroside and tyrosol accumulations. The present data also provided scientific basis for understanding complex interaction between endophytic fungus and R. crenulata.

This study aimed to improve rosmarinic acid (RA) production in the whole plant culture of Solenostemon scutellarioides through elicitation with phytopathogenic fungi. Amongst selected fungi, Aternaria alternata caused significant elevation (p

Disturbances in amino acid metabolism are increasingly recognized as being associated with, and serving as prognostic markers for chronic human diseases, such as cancer or type 2 diabetes. In the current study, a quantitative metabolomics profiling strategy revealed global impairment in amino acid metabolism in mice deleted for the transcriptional coactivator steroid receptor coactivator (SRC)-1. Aberrations were hepatic in origin, because selective reexpression of SRC-1 in the liver of SRC-1 null mice largely restored amino acids concentrations to normal levels. Cistromic analysis of SRC-1 binding sites in hepatic tissues confirmed a prominent influence of this coregulator on transcriptional programs regulating amino acid metabolism. More specifically, SRC-1 markedly impacted tyrosine levels and was found to regulate the transcriptional activity of the tyrosine aminotransferase (TAT) gene, which encodes the rate-limiting enzyme of tyrosine catabolism. Consequently, SRC-1 null mice displayed low TAT expression and presented with hypertyrosinemia and corneal alterations, 2 clinical features observed in the human syndrome of TAT deficiency. A heterozygous missense variant of SRC-1 (p.P1272S) that is known to alter its coactivation potential, was found in patients harboring idiopathic tyrosinemia-like disorders and may therefore represent one risk factor for their clinical symptoms. Hence, we reinforce the concept that SRC-1 is a central factor in the fine orchestration of multiple pathways of intermediary metabolism, suggesting it as a potential therapeutic target that may be exploitable in human metabolic diseases and cancer.

Rosmarinic acid (RA) and its derivants are medicinal compounds that comprise the active components of several therapeutics. We isolated and characterised a tyrosine aminotransferase of Prunella vulgaris (PvTAT). Deduced PvTAT was markedly homologous to other known/putative plant TATs. Cytoplasmic localisation of PvTAT was observed in tobacco protoplasts. Recombinantly expressed and purified PvTAT had substrates preference for L-tyrosine and phenylpyruvate, with apparent Km of 0.40 and 0.48 mM, and favoured the conversion of tyrosine to 4-hydroxyphenylpyruvate. In vivo activity was confirmed by functional restoration of the Escherichia coli tyrosine auxotrophic mutant DL39. Agrobacterium rhizogenes-mediated antisense/sense expression of PvTAT in hairy roots was used to evaluate the contribution of PvTAT to RA synthesis. PvTAT were reduced by 46–95% and RA were decreased by 36–91% with low catalytic activity in antisense transgenic hairy root lines; furthermore, PvTAT were increased 0.77–2.6-fold with increased 1.3–1.8-fold RA and strong catalytic activity in sense transgenic hairy root lines compared with wild-type counterparts. The comprehensive physiological and catalytic evidence fills in the gap in RA-producing plants which didn’t provide evidence for TAT expression and catalytic activities in vitro and in vivo. That also highlights RA biosynthesis pathway in P. vulgaris and provides useful information to engineer natural products.

Gene expression analysis using reverse transcription quantitative real-time PCR (RT-qPCR) requires the use of reference gene(s) in the target species. The long yellow daylily, Hemerocallis citrina Baroni. is rich in beneficial secondary metabolites and is considered as a functional vegetable. It is widely cultivated and consumed in East Asian countries. However, reference genes for use in RT-qPCR in H. citrina are not available. In the present study, six potential reference genes, actin (ACT), AP-4 complex subunit (AP4), tubulin (TUB), ubiquitin (UBQ), 18S and 60S ribosomal RNA, were selected and their expression stability in different developmental stages, organs and accessions was evaluated using four statistical software packages (geNorm, NormFinder, BestKeeper, and RefFinder). For commercial flower buds of different landraces, the combination of 60S, TUB, and AP4 was appropriate whereas ACT and 60S was suitable for normalization of different organs. In addition, AP4 exhibited the most stable expression in flower buds among different developmental stages. UBQ was less stable than the other reference genes under the experimental conditions except under different organs was 18S. The relative expression levels of two genes, primary-amine oxidase (HcAOC3) and tyrosine aminotransferase (HcTAT) which play important roles in alkaloid biosynthesis were also examined in different organs of the 'Datong' landrace, which further confirmed the results of selected reference genes. This is the first report to evaluate the stability of reference genes in the long yellow daylily that can serve as a foundation for RT-qPCR analysis of gene expression in this species.

Trypanosoma cruzi, the etiological agent of Chagas disease, lacks genes that encode canonical branched-chain aminotransferases. However, early studies showed that when epimastigotes were grown in the presence of 14C1-DL-leucine, the label was incorporated into various intermediates. More recently, our studies provided evidence that T. cruzi epimastigotes display a single ATP-dependent and saturable transport system that enables epimastigotes to uptake branched-chain amino acids (BCAAs) from the culture media. To extend our knowledge of the first step of BCAA catabolism, the ability of this parasite′s non-canonical broad specificity aminotransferases, such as tyrosine aminotransferase (TAT) and aspartate aminotransferase (ASAT), to transaminate these amino acids was investigated. Indeed, our results show that TAT and ASAT utilize BCAAs as substrates; however, both enzymes differ in their catalytic competence in utilizing these amino donors. For instance, ASAT transaminates isoleucine nearly 10-fold more efficiently than does TAT. This unique characteristic of TAT and ASAT allows to explain how BCAAs can be oxidized in the absence of a BCAA transaminase in T. cruzi. This article is protected by copyright. All rights reserved.

BACKGROUND: Tyrosinemia type II, also known as Richner-Hanhart Syndrome, is an extremely rare autosomal recessive disorder, caused by mutations in the gene encoding hepatic cytosolic tyrosine aminotransferase, leading to the accumulation of tyrosine and its metabolites which cause ocular and skin lesions, that may be accompanied by neurological manifestations, mostly intellectual disability. AIMS: To update disease-causing mutations and current clinical knowledge of the disease. MATERIALS AND METHODS: Genetic and clinical information were obtained from a collection of both unreported and previously reported cases. RESULTS: We report 106 families, represented by 143 individuals, carrying a total of 36 genetic variants, 11 of them not previously known to be associated with the disease. Variants include 3 large deletions, 21 non-synonymous and 5 nonsense amino-acid changes, 5 frameshifts and 2 splice variants. We also report 5 patients from Gran Canaria, representing the largest known group of unrelated families sharing the same P406L mutation. CONCLUSIONS: Data analysis did not reveal a genotype-phenotype correlation, but stressed the need of early diagnosis: All patients improved the oculocutaneous lesions after dietary treatment but neurological symptoms prevailed. The discovery of founder mutations in isolated populations, and the benefits of early intervention, should increase diagnostic awareness in newborns.

Classic glucocorticoids that have outstanding anti-inflammatory effects are still widely prescribed for the treatment of various inflammatory and autoimmune diseases. Conversely, glucocorticoids cause numerous unwanted side effects, particularly systemically dosed glucocorticoids. Therefore, selective glucocorticoid receptor modulator (SGRM), which maintains beneficial anti-inflammatory effects while reducing the occurrence of side effects, is one of the most anticipated drugs. However, there have been no SGRMs marketed to date. The assumption is that there are two major mechanisms of action of glucocorticoids via glucocorticoid receptors, transrepression (TR) and transactivation (TA). In general, the anti-inflammatory effects of glucocorticoids are mostly mediated through TR, while the side effects associated with glucocorticoids are largely caused by TA. We started to evaluate novel orally available SGRMs that maintain anti-inflammatory effects while minimizing adverse effects by favoring TR over TA. Based on this evaluation, we discovered JTP-117968, (4b'S,7'R,8a'S)-4b'-benzyl-7'-hydroxy-N-(2-methylpyridin-3-yl)-7'-(trifluoromethyl)-4b',6',7',8',8a',10'-hexahydro-5'H-spiro[cyclopropane-1,9'-phenanthrene]-2'-carboxamide, a non-steroidal SGRM. JTP-117968 has partial TR activity, but exhibits extremely low TA activity. The maximum TR efficacy of JTP-117968 was comparable to its structural analogue, PF-802, (4bS,7R,8aR)-4b-Benzyl-7-hydroxy-N-(2-methylpyridin-3-yl)-7-(trifluoromethyl)-4b,5,6,7,8,8a,9,10-octahydrophenanthrene-2-carboxamide, which is the active form of Fosdagrocorat that has been developed clinically as a first-in-class orally available SGRM. Remarkably, the TA activity of JTP-117968 was much weaker than PF-802 not only in in vitro assays, but also in in vivo mice experiments. These findings indicate that JTP-117968 exhibits improved TR/TA dissociation because the compound has significantly lower TA activity compared with an already reported SGRM. Therefore, JTP-117968 is expected to be a useful compound for evaluating ideal SGRMs in the future.

Mutations in the tyrosine aminotransferase gene have been identified to cause tyrosinemia type II which is inherited in an autosomal recessive manner. Studies have demonstrated that an excessive production of ROS can lead to reactions with macromolecules, such as DNA, lipids, and proteins. Considering that the L-tyrosine may promote oxidative stress, the main objective of this study was to investigate the in vivo effects of L-tyrosine on DNA damage determined by the alkaline comet assay, in brain and blood of rats. In our acute protocol, Wistar rats (30 days old) were killed 1 h after a single intraperitoneal L-tyrosine injection (500 mg/kg) or saline. For chronic administration, the animals received two subcutaneous injections of L-tyrosine (500 mg/kg, 12-h intervals) or saline administered for 24 days starting at postnatal day (PD) 7 (last injection at PD 31), 12 h after the last injection, the animals were killed by decapitation. We observed that acute administration of L-tyrosine increased DNA damage frequency and damage index in cerebral cortex and blood when compared to control group. Moreover, we observed that chronic administration of L-tyrosine increased DNA damage frequency and damage index in hippocampus, striatum, cerebral cortex and blood when compared to control group. In conclusion, the present work demonstrated that DNA damage can be encountered in brain from animal models of hypertyrosinemia, DNA alterations may represent a further means to explain neurological dysfunction in this inherited metabolic disorder and to reinforce the role of oxidative stress in the pathophysiology of tyrosinemia type II.

Identification of residues responsible for functional specificity in enzymes is a challenging and important problem in protein chemistry. Active site residues are generally easy to identify, but residues outside the active site are also important to catalysis and their identities and roles are more difficult to determine. We report a method based on analysis of multiple sequence alignments, embodied in our program Janus, for predicting mutations required to interconvert structurally related but functionally distinct enzymes. Conversion of aspartate aminotransferase into tyrosine aminotransferase is demonstrated and compared to previous efforts. Incorporation of 35 predicted mutations resulted in an enzyme with the desired substrate specificity but low catalytic activity. A single round of DNA back-shuffling with wild type aspartate aminotransferase on this variant generated mutants with tyrosine aminotransferase activities better than those previously realized from rational design or directed evolution. Methods such as this, coupled with computational modeling, may prove invaluable in furthering our understanding of enzyme catalysis and engineering.

To investigate roles of nitric oxide (NO) signal in accumulations of phenolic acids in abscisic.acid (ABA)-induced Salvia miltiorrhiza hairy roots, S. miltiorrhiza hairy roots were treated with different concentrations of sodium nitroprusside (SNP)-an exogenous NO donor, for 6 days, and contents of phenolic acids in the hairy roots are determined. Then with treatment of ABA and NO scavenger (2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1- oxyl-3-oxide, c-PTIO) or NO synthase inhibitor (NG-nitro-L-arginine methyl ester, L-NAME), contents of phenolic acids and expression levels of three key genes involved in phenolic acids biosynthesis were detected. Phenolic acids production in S. miltiorrhiza hairy roots was most significantly improved by 100 µmoL/L SNP. Contents of RA and salvianolic acid B increased by 3 and 4 folds. ABA significantly improved transcript levels of PAL (phenylalanine ammonia lyase), TAT (tyrosine aminotransferase) and RAS (rosmarinic acid synthase), and increased phenolic acids accumulations. However, with treatments of ABA+c-PTIO or ABA+L-NAME, accumulations of phenolic acids and expression levels of the three key genes were significantly inhibited. Both NO and ABA can increase accumulations of phenolic acids in S. miltiorrhiza hairy roots. NO signal probably mediates the ABA-induced phenolic acids production.

none 5 no This research was supported by funds from the University of Bologna (RFO2014) to S.B.and from the University of Urbino (RFO 2014) to V.S.. Quinoa (Chenopodium quinoa Willd), an ancient Andean seed crop, exhibits exceptional nutritional properties and resistance to abiotic stress. The species' tolerance to heavy metals has, however, not yet been investigated nor its ability to take up and translocate chromium (Cr). This study aimed to investigate the metabolic adjustments occurring upon exposure of quinoa to several concentrations (0.01–5mM) of CrCl3. Young hydroponically grown plants were used to evaluate Cr uptake, growth, oxidative stress, and other biochemical parameters three and/or seven days after treatment. Leaves accumulated the lowest amounts of Cr, while roots and stems accumulated the most at low and at high metal concentrations, respectively. Fresh weight and photosynthetic pigments were reduced only by the higher Cr(III) doses. Substantially increased lipid peroxidation, hydrogen peroxide, and proline levels were observed only with 5mM Cr(III). Except for a significant decrease at day 7 with 5mM Cr(III), total polyphenols and flavonoids maintained control levels in Cr(III)-treated plants, whereas antioxidant activity increased in a dose-dependent manner. Maximum polyamine accumulation was observed in 1mM CrCl3-treated plants. Even though α- and γ-tocopherols also showed enhanced levels only with the 1mM concentration, tyrosine aminotransferase (TAT, EC 2.6.1.5) activity increased under Cr(III) treatment in a dose- and time-dependent manner. Taken together, results suggest that polyamines, tocopherols, and TAT activity could contribute to tolerance to 1mM Cr(III), but not to the highest concentration that, instead, generated oxidative stress. open Scoccianti, Valeria; Bucchini, Anahi E.; Iacobucci, Marta; Ruiz, Karina B.; Biondi, Stefania Scoccianti, Valeria; Bucchini, Anahi E.; Iacobucci, Marta; Ruiz, Karina B.; Biondi, Stefania

Blood-feeding arthropods are vectors of infectious diseases such as dengue, Zika, Chagas disease, and malaria [1], and vector control is essential to limiting disease spread. Because these arthropods ingest very large amounts of blood, a protein-rich meal, huge amounts of amino acids are produced during digestion. Previous work on Rhodnius prolixus, a vector of Chagas disease, showed that, among all amino acids, only tyrosine degradation enzymes were overexpressed in the midgut compared to other tissues [2]. Here we demonstrate that tyrosine detoxification is an essential trait in the life history of blood-sucking arthropods. We found that silencing Rhodnius tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD), the first two enzymes of the phenylalanine/tyrosine degradation pathway, caused the death of insects after a blood meal. This was confirmed by using the HPPD inhibitor mesotrione, which selectively killed hematophagous arthropods but did not affect non-hematophagous insects. In addition, mosquitoes and kissing bugs died after feeding on mice that had previously received a therapeutic effective oral dose (1 mg/kg) of nitisinone, another HPPD inhibitor used in humans for the treatment of tyrosinemia type I [3]. These findings indicate that HPPD (and TAT) can be a target for the selective control of blood-sucking disease vector populations. Because HPPD inhibitors are extensively used as herbicides and in medicine, these compounds may provide an alternative less toxic to humans and more environmentally friendly than the conventional neurotoxic insecticides that are currently used, with the ability to affect only hematophagous arthropods.

MicroRNAs (miRNAs) have a fundamental role in diabetic heart failure. The cardioprotective miRNA-133a (miR-133a) is downregulated, and contractility is decreased in diabetic hearts. Norepinephrine (NE) is a key catecholamine that stimulates contractility by activating β-adrenergic receptors (β-AR). NE is synthesized from tyrosine by the rate-limiting enzyme, tyrosine hydroxylase (TH), and tyrosine is catabolized by tyrosine aminotransferase (TAT). However, the cross talk/link between TAT and TH in the heart is unclear. To determine whether miR-133a plays a role in the cross talk between TH and TAT and regulates contractility by influencing NE biosynthesis and/or β-AR levels in diabetic hearts, Sprague-Dawley rats and miR-133a transgenic (miR-133aTg) mice were injected with streptozotocin to induce diabetes. The diabetic rats were then treated with miR-133a mimic or scrambled miRNA. Our results revealed that miR-133a mimic treatment improved the contractility of the diabetic rat’s heart concomitant with upregulation of TH, cardiac NE, β-AR, and downregulation of TAT and plasma levels of NE. In miR-133aTg mice, cardiac-specific miR-133a overexpression prevented upregulation of TAT and suppression of TH in the heart after streptozotocin was administered. Moreover, miR-133a overexpression in CATH.a neuronal cells suppressed TAT with concomitant upregulation of TH, whereas knockdown and overexpression of TAT demonstrated that TAT inhibited TH. Luciferase reporter assay confirmed that miR-133a targets TAT. In conclusion, miR-133a controls the contractility of diabetic hearts by targeting TAT, regulating NE biosynthesis, and consequently, β-AR and cardiac function.

Tyrosine levels are abnormally elevated in tissues and physiological fluids of patients with inborn errors of tyrosine catabolism, especially in tyrosinemia type II, which is caused by deficiency of tyrosine aminotransferase and provokes eyes, skin, and central nervous system disturbances. Considering that the mechanisms of brain damage in these disorders are poorly known, in this study, we investigated the in vivo and in vitro effects of tyrosine on some parameters of energy metabolism in cerebral cortex of 14-day-old Wistar rats. We observed that 2 mM tyrosine inhibited in vitro the pyruvate kinase (PK) activity and that this inhibition was prevented by 1 mM reduced glutathione with 30, 60, and 90 min of preincubation. Moreover, administration of tyrosine methyl ester (TME) (0.5 mg/g of body weight) decreased the activity of PK and this reduction was prevented by pre-treatment with creatine (Cr). On the other hand, tyrosine did not alter adenylate kinase (AK) activity in vitro, but administration of TME enhanced AK activity not prevented by Cr pre-treatment. Finally, TME administration decreased the activity of CK from cytosolic and mitochondrial fractions and this diminution was prevented by Cr pre-treatment. The results suggest that tyrosine alters essential sulfhydryl groups necessary for CK and PK functions, possibly through oxidative stress. In case this also occurs in the patients, it is possible that energy metabolism alterations may contribute, along with other mechanisms, to the neurological dysfunction of hypertyrosinemias.

BACKGROUND: The mode of action of the grass herbicides cinmethylin and 5-benzyloxymethyl-1,2-isoxazolines substituted with methylthiophene (methiozolin) or pyridine (ISO1, ISO2) was investigated. RESULTS: Physiological profiling using a series of biotests and metabolic profiling in treated duckweed (Lemna paucicostata L.) suggested a common mode of action for the herbicides. Symptoms of growth inhibition and photobleaching of new fronds in Lemna were accompanied with metabolite changes indicating an upregulation of shikimate and tyrosine metabolism, paralleled by decreased plastoquinone and carotenoid synthesis. Supplying Lemna with 10 µM of 4-hydroxyphenylpyruvate (4-HPP) reversed phytotoxic effects of cinmethylin and isoxazolines to a great extent, whereas the addition of L-tyrosine was ineffective. It was hypothesised that the herbicides block the conversion of tyrosine to 4-HPP, catalysed by tyrosine aminotransferase (TAT), in the prenylquinone pathway which provides plastoquinone, a cofactor of phytoene desaturase in carotenoid synthesis. Accordingly, enhanced resistance to ISO1 treatment was observed in Arabidopsis thaliana L. mutants, which overexpress the yeast prephenate dehydrogenase in plastids as a TAT bypass. In addition, the herbicides were able to inhibit TAT7 activity in vitro for the recombinant enzyme of A. thaliana. CONCLUSION: The results suggest that TAT7 or another TAT isoenzyme is the putative target of the herbicides. Copyright © 2011 Society of Chemical Industry

The existence of low levels of intersubunit communication in homooligomeric enzymes is often difficult to discover, as the identical active sites cannot be probed individually to dissect their interdependent contributions. The homodimeric paralogs, E. coli aspartate- (AATase) and tyrosine aminotransferase (TATase), have not been demonstrated to show allostery. To address this question, we engineered a hybrid aminotransferase containing two distinct catalytic pockets: an AATase and a TATase site. The TATase/AATase hybrid was constructed by grafting an engineered TATase active site into one of the catalytic pockets of E. coli AATase. Each active site conserves its specific catalytic and inhibitor binding properties, and the hybrid catalyzes simultaneously each aminotransferase reaction at the respective site. Importantly, association of a selective inhibitor into one of the catalytic pockets decreases the activity of the second active site by up to 25%, thus proving unequivocally the existence of allosteric communication between active sites. The procedure may be applicable to other homologous sets of enzymes.

Structurally novel 5H-chromeno[2,3-b]pyridine (azaxanthene) selective glucocorticoid receptor (GR) modulators have been identified. A screening paradigm utilizing cellular assays of GR-mediated transrepression of proinflammatory transcription factors and transactivation of GR-dependent genes combined with three physiologically relevant assays of cytokine induction in human whole blood has allowed for the identification of high affinity, selective GR ligands that display a broad range of pharmacological profiles. Agonist efficacy in reporter assays can be tuned by halogenation of a pendent phenyl ring and correlates well with efficacy for cytokine inhibition in human whole blood. A hypothetical binding mode is proposed, invoking an expanded ligand binding pocket resembling that of arylpyrazole-bound GR structures. Two compounds of close structural similarity (35 and 37; BMS-776532 and BMS-791826, respectively) have been found to maintain distinct and consistent levels of partial agonist efficacy across several assays, displaying anti-inflammatory activity comparable to that of prednisolone 2 in suppressing cytokine production in whole blood and in rodent models of acute and chronic inflammation.

The present work investigated the effects of salicylic acid (SA) on the accumulation of phenolic compounds and the activities of PAL, TAT, SOD, CAT and POD enzymes in the Salvia miltiorrhiza cell culture. When SA is applied to the cell culture, phenolic compounds will increase and PAL, TAT, SOD, CAT, and POD enzymes will become more active. The accumulations of phenolic compounds and the PAL activity were stimulated 8h after the treatment with SA. The TAT activity was stimulated after 48 h. The resulting antioxidative enzymes' activities were greatly improved. SA elicitation on the phenolic acid accumulation was depended upon the application dosage and the time-duration. The suitable SA concentration for eliciting phenolic compound accumulations was 6.25-22.5mg/L. The elicitation effect of SA on phenolic compound accumulations correlated with the PAL activity, but not with the TAT activity. This indicates that PAL may be the key enzyme for the biosynthesis of salvianolic acid B and caffeic acid. The raised PAL activity leads to the improvement of the quantity of phenolic compounds. This could be of particular significance by using plant cell culture systems for biotechnological production of plant secondary metabolites such as salvianolic acid B and caffeic acid.

Compound A (CpdA), a dissociated glucocorticoid receptor modulator, decreases corticosteroid-binding globulin (CBG), adrenocorticotropic hormone (ACTH), and luteneinizing hormone levels in rats. Whether this is due to transcriptional regulation by CpdA is not known. Using promoter reporter assays we show that CpdA, like dexamethasone (Dex), directly transrepresses these genes. Results using a rat Cbg proximal-promoter reporter construct in BWTG3 and HepG2 cell lines support a glucocorticoid receptor (GR)-dependent transrepression mechanism for CpdA. However, CpdA, unlike Dex, does not result in transactivation via glucocorticoid-responsive elements within a promoter reporter construct even when GR is co-transfected. The inability of CpdA to result in transactivation via glucocorticoid-responsive elements is confirmed on the endogenous tyrosine aminotransferase gene, whereas transrepression ability is confirmed on the endogenous CBG gene. Consistent with a role for CpdA in modulating GR activity, whole cell binding assays revealed that CpdA binds reversibly to the GR, but with lower affinity than Dex, and influences association of [(3)H]Dex, but has no effect on dissociation. In addition, like Dex, CpdA causes nuclear translocation of the GR, albeit to a lesser degree. Several lines of evidence, including fluorescence resonance energy transfer, co-immunoprecipitation, and nuclear immunofluorescence studies of nuclear localization-deficient GR show that CpdA, unlike Dex, does not elicit ligand-induced GR dimerization. Comparison of the behavior of CpdA in the presence of wild type GR to that of Dex with a dimerization-deficient GR mutant (GR(dim)) strongly supports the conclusion that loss of dimerization is responsible for the dissociated behavior of CpdA.