Transcriptional heterogeneity of catabolic genes on the plasmid pCAR1 causes host-specific carbazole degradation.

To elucidate why plasmid-borne catabolic ability differs among host bacteria, we assessed the expression dynamics of the Pant promoter on the carbazole-degradative conjugative plasmid pCAR1 in Pseudomonas putida KT2440(pCAR1) (hereafter, KTPC) and Pseudomonas resinovorans CA10. The Pant promoter regulates the transcription of both the car and ant operons, which are responsible for converting carbazole into anthranilate and anthranilate into catechol, respectively. In the presence of anthranilate, transcription of the Pant promoter is induced by the AraC/XylS family regulator AntR, encoded on pCAR1. A reporter cassette containing the Pant promoter followed by gfp was inserted into the chromosomes of KTPC and CA10. After adding anthranilate, GFP expression in the population of CA10 showed an unimodal distribution, whereas a small population with low GFP fluorescence intensity appeared for KTPC. CA10 has a gene, antRCA, that encodes an iso-functional homolog of AntR on its chromosome. When antRCA was disrupted, a small population with low GFP fluorescence intensity appeared. In contrast, overexpression of pCAR1-encoded AntR in KTPC resulted in unimodal expression under the Pant promoter. These results suggest that the expression of pCAR1-encoded AntR is insufficient to ameliorate the stochastic expression of the Pant promoter. Raman spectra of single cells collected using deuterium-labeled carbazole showed that the C-D Raman signal exhibited greater variability for KTPC than CA10. These results indicate that heterogeneity at the transcriptional level of the Pant promoter due to insufficient AntR availability causes fluctuations in the pCAR1-borne carbazole-degrading capacity of host bacterial cells.IMPORTANCEHorizontally acquired genes increase the competitiveness of host bacteria under selective conditions, although unregulated expression of foreign genes may impose fitness costs. The "appropriate" host for a plasmid is empirically known to maximize the expression of plasmid-borne traits. In the case of pCAR1-harboring Pseudomonas strains, P. resinovorans CA10 exhibits strong carbazole-degrading capacity, whereas P. putida KT2440 harboring pCAR1 exhibits low degradation capacity. Our results suggest that a chromosomally encoded transcription factor affects transcriptional and metabolic fluctuations in host cells, resulting in different carbazole-degrading capacities as a population. This study may provide a clue for determining appropriate hosts for a plasmid and for regulating the expression of plasmid-borne traits, such as the degradation of xenobiotics and antibiotic resistance.

The biochemically defined super relaxed state of myosin-A paradox.

The biochemical SRX (super-relaxed) state of myosin has been defined as a low ATPase activity state. This state can conserve energy when the myosin is not recruited for muscle contraction. The SRX state has been correlated with a structurally defined ordered (versus disordered) state of muscle thick filaments. The two states may be linked via a common interacting head motif (IHM) where the two heads of heavy meromyosin (HMM), or myosin, fold back onto each other and form additional contacts with S2 and the thick filament. Experimental observations of the SRX, IHM, and the ordered form of thick filaments, however, do not always agree, and result in a series of unresolved paradoxes. To address these paradoxes, we have reexamined the biochemical measurements of the SRX state for porcine cardiac HMM. In our hands, the commonly employed mantATP displacement assay was unable to quantify the population of the SRX state with all data fitting very well by a single exponential. We further show that mavacamten inhibits the basal ATPases of both porcine ventricle HMM and S1 (Ki, 0.32 and 1.76 µM respectively) while dATP activates HMM cooperatively without any evidence of an SRX state. A combination of our experimental observations and theories suggests that the displacement of mantATP in purified proteins is not a reliable assay to quantify the SRX population. This means that while the structurally defined IHM and ordered thick filaments clearly exist, great care must be employed when using the mantATP displacement assay.

Uptake, Translocation, and Distribution of Cyantraniliprole in a Wheat Planting System.

Cyantraniliprole uptake, translocation, and distribution in wheat plants grown in hydroponics and soil conditions were investigated. The hydroponics experiment indicated that cyantraniliprole was prone to be absorbed by wheat roots mainly through the apoplastic pathway and predominately distributed in the cell-soluble fraction (81.4-83.6%) and ultimately transferred upward to leaves (TFleave/stem = 4.84 > TFstem/root = 0.67). In wheat-soil systems, the uptake of cyantraniliprole was similar to that in hydroponics. The accumulation of cyantraniliprole in wheat tissues was mainly affected by the content of soil organic matter and clay, resulting in the increased adsorption of cyantraniliprole onto soils (R2 > 0.991, P < 0.01), and was positively related to the concentration of cyantraniliprole in soil pore water (R2 > 0.991, P < 0.001). Besides, the absorption of cyantraniliprole by wheat was predicted well by the partition-limited model. These results increased our understanding of the absorption and accumulation of cyantraniliprole in wheat and were also helpful for guiding the practical application and risk evaluation of cyantraniliprole.

Structure, mechanism and inhibition of anthranilate phosphoribosyltransferase.

Anthranilate phosphoribosyltransferase catalyses the second reaction in the biosynthesis of tryptophan from chorismate in microorganisms and plants. The enzyme is homodimeric with the active site located in the hinge region between two domains. A range of structures in complex with the substrates, substrate analogues and inhibitors have been determined, and these have provided insights into the catalytic mechanism of this enzyme. Substrate 5-phospho-d-ribose 1-diphosphate (PRPP) binds to the C-terminal domain and coordinates to Mg2+, in a site completed by two flexible loops. Binding of the second substrate anthranilate is more complex, featuring multiple binding sites along an anthranilate channel. This multi-modal binding is consistent with the substrate inhibition observed at high concentrations of anthranilate. A series of structures predict a dissociative mechanism for the reaction, similar to the reaction mechanisms elucidated for other phosphoribosyltransferases. As this enzyme is essential for some pathogens, efforts have been made to develop inhibitors for this enzyme. To date, the best inhibitors exploit the multiple binding sites for anthranilate. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Anthranilic Acid Accumulation in Saccharomyces cerevisiae Induced by Expression of a Nonribosomal Peptide Synthetase Gene from Paecilomyces cinnamomeus BCC 9616.

Heterologous expression of nrps33, a nonribosomal peptide synthetase gene, from Paecilomyces cinnamomeus BCC 9616 in Saccharomyces cerevisiae unexpectedly resulted in the accumulation of anthranilic acid, an intermediate in tryptophan biosynthesis. Based on transcriptomic and real-time quantitative polymerase chain reaction (RT-qPCR) results, expression of nrps33 affected the transcription of tryptophan biosynthesis genes especially TRP1 which is also the selectable auxotrophic marker for the expression vector used in this work. The product of nrps33 could inhibit the activity of Trp4 involved in the conversion of anthranilate to N-(5'-phosphoribosyl)anthranilate and therefore caused the accumulation of anthranilic acid. This accumulation could in turn result in down-regulation of downstream tryptophan biosynthesis genes. Anthranilic acid is typically produced by chemical synthesis and has been used as a substrate for synthesising bioactive compounds including commercial drugs; our results could provide a new biological platform for production of this compound.

Improving Insecticidal Activity of Chlorantraniliprole by Replacing the Chloropyridinyl Moiety with a Substituted Cyanophenyl Group.

Insect ryanodine receptors (RyRs) are molecular targets of the anthranilic diamide insecticides. In the present study, a new series of anthranilic diamides containing a cyanophenyl pyrazole moiety were rationally designed by active-fragment assembly and computer-aided design using the 3D structure of Plutella xylostella RyRs as a receptor and chlorantraniliprole as a ligand. Most of the titled compounds showed good toxicity against Mythimna separate, P. xylostella, and Spodoptera frugiperda. Compounds CN06, CN11, and CN16 with corresponding LC50 values of 0.15, 0.29, and 0.52 mg·L-1, respectively, against M. separate showed comparable activity to that of chlorantraniliprole (0.13 mg·L-1). Surprisingly, CN06, CN11, and CN16 with corresponding LC50 values of 1.6 × 10-5, 3.0 × 10-5, and 2.8 × 10-5 mg·L-1, respectively, against P. xylostella were at least 5-fold more active than chlorantraniliprole (1.5 × 10-4 mg·L-1). In the case of S. frugiperda, CN06, CN11, and CN16 had good potency but lower than chlorantraniliprole in terms of LC50 values (0.58, 0.54, and 0.56 mg·L-1 versus 0.31 mg·L-1). Molecular docking of CN06 and chlorantraniliprole to P. xylostella RyRs validated the molecular design, and the calcium imaging technique further proved the potential target of CN06 as RyRs. Compounds CN06, CN11, and CN16 could be more effective than chlorantraniliprole in targeting the resistant RyR mutants of S. frugiperda (G4891E, I4734M) through the binding mode and energy obtained by molecular docking. Density functional theory calculations (DFT) and electrostatic potential (ESP) studies gave the structure-activity relationship. Compounds CN06, CN11, and CN16 could be used as potent insecticide leads for further optimization.

Uptake, translocation and distribution of cyantraniliprole in rice planting system.

While cyantraniliprole has been frequently used in rice fields, knowledge of the uptake, translocation and distribution of cyantraniliprole in rice planting systems is still largely unexplored. Plant uptake is a crucial factor in determining how cyantraniliprole moves through the food chain. Understanding the uptake, translocation and distribution of cyantraniliprole in rice planting system is essential to predicting its accumulation in rice and potential human exposure. Herein, the uptake process of cyantraniliprole in a hydroponic-rice system was systematically investigated. Results showed that cyantraniliprole was easily absorbed by rice roots via a passive diffusion process through the apoplastic pathway and then translocated upward through the xylem, but its acropetal translocation was limited. Cyantraniliprole in shoots can also be downward translocated through the phloem, although only to a limited extent, showing rice plants' weak phloem movement capacity. Furthermore, cyantraniliprole had a short half-life in sediment-water system and dissipated faster in anaerobic than aerobic conditions. At the equilibrium stage of a sediment-water system, cyantraniliprole is preferentially partitioned to the solid phase. Our study provides a systematic insight into the uptake, translocation and distribution of cyantraniliprole in the rice planting system, which is very helpful for better field cyantraniliprole application and environmental risk assessment.

Dynamic flux regulation for high-titer anthranilate production by plasmid-free, conditionally-auxotrophic strains of Pseudomonas putida.

Anthranilate, an intermediate of the shikimate pathway, is a high-value aromatic compound widely used as a precursor in the production of dyes, fragrances, plastics and pharmaceuticals. Traditional strategies adopted for microbial anthranilate production rely on the implementation of auxotrophic strains-which requires aromatic amino acids or complex additives to be supplemented in the culture medium, negatively impacting production costs. In this work, we engineered the soil bacterium Pseudomonas putida for high-titer, glucose-dependent anthranilate production by repurposing elements of the Esa quorum sensing (QS) system of Pantoea stewartii. The PesaS promoter mediated a self-regulated transcriptional response that effectively knocked-down the expression of the trpDC genes. Next, we harnessed the synthetic QS elements to engineer a growth-to-anthranilate production switch. The resulting plasmid-free P. putida strain produced the target compound at 3.8 ± 0.3 mM in shaken-flask cultures after 72 h-a titer >2-fold higher than anthranilate levels reported thus far. Our results highlight the value of dynamic flux regulation for the production of intermediate metabolites within highly-regulated routes (such as the shikimate pathway), thereby circumventing the need of expensive additives.

Anthranilate Acts as a Signal to Modulate Biofilm Formation, Virulence, and Antibiotic Tolerance of Pseudomonas aeruginosa and Surrounding Bacteria.

Anthranilate is a diffusible molecule produced by Pseudomonas aeruginosa and accumulates as P. aeruginosa grows. Anthranilate is an important intermediate for the synthesis of tryptophan and the Pseudomonas quinolone signal (PQS), as well as metabolized by the anthranilate dioxygenase complex (antABC operon products). Here we demonstrate that anthranilate is a key factor that modulates the pathogenicity-related phenotypes of P. aeruginosa and other surrounding bacteria in the environment, such as biofilm formation, antibiotic tolerance, and virulence. We found that the anthranilate levels in P. aeruginosa cultures rapidly increased in the stationary phase and then decreased again, forming an anthranilate peak. Biofilm formation, antibiotic susceptibility, and virulence of P. aeruginosa were significantly altered before and after this anthranilate peak. In addition, these phenotypes were all modified by the mutation of antABC and exogenous addition of anthranilate. Anthranilate also increased the antibiotic susceptibility of other species of bacteria, such as Escherichia coli, Salmonella enterica, Bacillus subtilis, and Staphylococcus aureus. Before the anthranilate peak, the low intracellular anthranilate level was maintained through degradation from the antABC function, in which induction of antABC was also limited to a small extent. The premature degradation of anthranilate, due to its high levels, and antABC expression early in the growth phase, appears to be toxic to the cells. From these results, we propose that by generating an anthranilate peak as a signal, P. aeruginosa may induce some sort of physiological change in surrounding cells. IMPORTANCE Pseudomonas aeruginosa is a notorious pathogen with high antibiotic resistance, strong virulence, and ability to cause biofilm-mediated chronic infection. We found that these characteristics change profoundly before and after the time when anthranilate is produced as an "anthranilate peak". This peak acts as a signal that induces physiological changes in surrounding cells, decreasing their antibiotic tolerance and biofilm formation. This study is important in that it provides a new insight into how microbial signaling substances can induce changes in the pathogenicity-related phenotypes of cells in the environment. In addition, this study shows that anthranilate can be used as an adjuvant to antibiotics.

Anti-Inflammatory Anthranilate Analogue Enhances Autophagy through mTOR and Promotes ER-Turnover through TEX264 during Alzheimer-Associated Neuroinflammation.

Autophagy degrades impaired organelles and toxic proteins to maintain cellular homeostasis. Dysregulated autophagy is a pathogenic participant in Alzheimer's disease (AD) progression. In early-stage AD, autophagy is beneficially initiated by mild endoplasmic reticulum (ER) stress to alleviate cellular damage and inflammation. However, chronic overproduction of toxic Aß oligomers eventually causes Ca2+ dysregulation in the ER, subsequently elevating ER-stress and impairing autophagy. Our previous work showed that a novel anthranilate analogue (SI-W052) inhibited lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α and interleukin (IL)-6 on microglia. To investigate its mechanism of action, herein, we postulate that SI-W052 exhibits anti-inflammatory activity through ER-stress-mediated autophagy. We initially demonstrate that autophagy inhibits inflammation, but it becomes impaired during acute inflammation. SI-W052 significantly induces the conversion ratio of LC3 II/I and inhibits LPS-upregulated p-mTOR, thereby restoring impaired autophagy to modulate inflammation. Our signaling study further indicates that SI-W052 inhibits the upregulation of ER-stress marker genes, including Atf4 and sXbp1/tXbp1, explaining compound activity against IL-6. This evidence encouraged us to evaluate ER-stress-triggered ER-phagy using TEX264. ER-phagy mediates ER-turnover by the degradation of ER fragments to maintain homeostasis. TEX264 is an important ER-phagy receptor involved in ATF4-mediated ER-phagy under ER-stress. In our study, elevated TEX264 degradation is identified during inflammation; SI-W052 enhances TEX264 expression, producing a positive effect in ER-turnover. Our knockdown experiment further verifies the important role of TEX264 in SI-W052 activity against IL-6 and ER-stress. In conclusion, this study demonstrates that an anthranilate analogue is a novel neuroinflammation agent functioning through ER-stress-mediated autophagy and ER-phagy mechanisms.

Methyl and Isopropyl N-Methylanthranilates Affect Primary Macrophage Function - An Insight into the Possible Immunomodulatory Mode of Action.

To complement the knowledge on the anti-inflammatory activity of methyl and isopropyl N-methylanthranilates, two natural products with panacea-like properties, we investigated their effects on thioglycolate-elicited macrophages by evaluating macrophage ability to metabolize MTT, macrophage membrane function, and macrophage myeloperoxidase and phagocytic activities. Moreover, two additional aspects of the inflammatory response of these compounds, their inhibitory activity on xanthine oxidase and catalase, were studied. It was found that these two compounds regulate elicited macrophage functions, most probably by interfering with the function of cell membranes and changing the reducing cellular capacity or enzyme activity of macrophages. Nonetheless, no significant inhibitory action either towards xanthine oxidase or catalase was found, suggesting that the inhibition of these enzymes is not involved in the anti-inflammatory mode of action of these two esters.

Chimeric Investigations into the Diamide Binding Site on the Lepidopteran Ryanodine Receptor.

Alterations to amino acid residues G4946 and I4790, associated with resistance to diamide insecticides, suggests a location of diamide interaction within the pVSD voltage sensor-like domain of the insect ryanodine receptor (RyR). To further delineate the interaction site(s), targeted alterations were made within the same pVSD region on the diamondback moth (Plutella xylostella) RyR channel. The editing of five amino acid positions to match those found in the diamide insensitive skeletal RyR1 of humans (hRyR1) in order to generate a human-Plutella chimeric construct showed that these alterations strongly reduce diamide efficacy when introduced in combination but cause only minor reductions when introduced individually. It is concluded that the sites of diamide interaction on insect RyRs lie proximal to the voltage sensor-like domain of the RyR and that the main site of interaction is at residues K4700, Y4701, I4790 and S4919 in the S1 to S4 transmembrane domains.

Avenanthramide C as a novel candidate to alleviate osteoarthritic pathogenesis.

Osteoarthritis (OA) is a degenerative disorder that can result in the loss of articular cartilage. No effective treatment against OA is currently available. Thus, interest in natural health products to relieve OA symptoms is increasing. However, their qualities such as efficacy, toxicity, and mechanism are poorly understood. In this study, we determined the efficacy of avenanthramide (Avn)-C extracted from oats as a promising candidate to prevent OA progression and its mechanism of action to prevent the expression of matrix-metalloproteinases (MMPs) in OA pathogenesis. Interleukin-1 beta (IL-1ß), a proinflammatory cytokine as a main causing factor of cartilage destruction, was used to induce OAlike condition of chondrocytes in vitro. Avn-C restrained IL-1ß- mediated expression and activity of MMPs, such as MMP-3, -12, and -13 in mouse articular chondrocytes. Moreover, Avn-C alleviated cartilage destruction in experimental OA mouse model induced by destabilization of the medial meniscus (DMM) surgery. However, Avn-C did not affect the expression of inflammatory mediators (Ptgs2 and Nos) or anabolic factors (Col2a1, Aggrecan, and Sox9), although expression levels of these genes were upregulated or downregulated by IL-1ß, respectively. The inhibition of MMP expression by Avn-C in articular chondrocytes was mediated by p38 kinase and c-Jun N-terminal kinase (JNK) signaling, but not by ERK or NF-κB. Interestingly, Avn-C added with SB203580 and SP600125 as specific inhibitors of p38 kinase and JNK, respectively, enhanced its inhibitory effect on the expression of MMPs in IL-1ß treated chondrocytes. Taken together, these results suggest that Avn-C is an effective candidate to prevent OA progression and a natural health product to relieve OA pathogenesis. [BMB Reports 2021; 54(10): 528-533].

Combinatorial Assembly of Modular Glucosides via Carboxylesterases Regulates C. elegans Starvation Survival.

The recently discovered modular glucosides (MOGLs) form a large metabolite library derived from combinatorial assembly of moieties from amino acid, neurotransmitter, and lipid metabolism in the model organism C. elegans. Combining CRISPR-Cas9 genome editing, comparative metabolomics, and synthesis, we show that the carboxylesterase homologue Cel-CEST-1.2 is responsible for specific 2-O-acylation of diverse glucose scaffolds with a wide variety of building blocks, resulting in more than 150 different MOGLs. We further show that this biosynthetic role is conserved for the closest homologue of Cel-CEST-1.2 in the related nematode species C. briggsae, Cbr-CEST-2. Expression of Cel-cest-1.2 and MOGL biosynthesis are strongly induced by starvation conditions in C. elegans, one of the premier model systems for mechanisms connecting nutrition and physiology. Cel-cest-1.2-deletion results in early death of adult animals under starvation conditions, providing first insights into the biological functions of MOGLs.

Lead optimization to improve the antiviral potency of 2-aminobenzamide derivatives targeting HIV-1 Vif-A3G axis.

The viral infectivity factor (Vif)-apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G) axis has been recognized as a valid target for developing novel small-molecule therapies for acquired immune deficiency syndrome (AIDS) or for enhancing innate immunity against viruses. Our previous work reported the novel Vif antagonist 2-amino-N-(2-methoxyphenyl)-6-((4-nitrophenyl)sulfonyl)benzamide (2) with strong antiviral activity. In this work, through optimizations of ring C of 2, we discovered the more potent compound 6m with an EC50 of 0.07 µM in non-permissive H9 cells, reflecting an approximately 5-fold enhancement of antiviral activity compared to that of 2. Western blotting indicated that 6m more strongly suppressed the defensive protein Vif than 2 at the same concentration. Furthermore, 6m suppressed the replication of various clinical drug-resistant HIV strains (FI, NRTI, NNRTI, IN and PI) with relatively high efficacy. These results suggested that compound 6m is a more potent candidate for treating AIDS.

Synergistic Effect of Methyl Jasmonate and Abscisic Acid Co-Treatment on Avenanthramide Production in Germinating Oats.

The oat (Avena sativa L.) is a grain of the Poaceae grass family and contains many powerful anti-oxidants, including avenanthramides as phenolic alkaloids with anti-inflammatory, anti-oxidant, anti-itch, anti-irritant, and anti-atherogenic activities. Here, the treatment of germinating oats with methyl jasmonate (MeJA) or abscisic acid (ABA) resulted in 2.5-fold (582.9 mg/kg FW) and 2.8-fold (642.9 mg/kg FW) increase in avenanthramide content, respectively, relative to untreated controls (232.6 mg/kg FW). Moreover, MeJA and ABA co-treatment synergistically increased avenanthramide production in germinating oats to 1505 mg/kg FW. Individual or combined MeJA and ABA treatment increased the expression of genes encoding key catalytic enzymes in the avenanthramide-biosynthesis pathway, including hydroxycinnamoyl-CoA:hydrocyanthranilate N-hydroxycinnamoyl transferase (HHT). Further analyses showed that six AsHHT genes were effectively upregulated by MeJA or ABA treatment, especially AsHHT4 for MeJA and AsHHT5 for ABA, thereby enhancing the production of all three avenanthramides in germinating oats. Specifically, AsHHT5 exhibited the highest expression following MeJA and ABA co-treatment, indicating that AsHHT5 played a more crucial role in avenanthramide biosynthesis in response to MeJA and ABA co-treatment of germinating oats. These findings suggest that elicitor-mediated metabolite farming using MeJA and ABA could be a valuable method for avenanthramide production in germinating oats.

Avenanthramide Metabotype from Whole-Grain Oat Intake is Influenced by Faecalibacterium prausnitzii in Healthy Adults.

BACKGROUND: Oat has been widely accepted as a key food for human health. It is becoming increasingly evident that individual differences in metabolism determine how different individuals benefit from diet. Both host genetics and the gut microbiota play important roles on the metabolism and function of dietary compounds. OBJECTIVES: To investigate the mechanism of individual variations in response to whole-grain (WG) oat intake. METHODS: We used the combination of in vitro incubation assays with human gut microbiota, mouse and human S9 fractions, chemical analyses, germ-free (GF) mice, 16S rRNA sequencing, gnotobiotic techniques, and a human feeding study. RESULTS: Avenanthramides (AVAs), the signature bioactive polyphenols of WG oat, were not metabolized into their dihydro forms, dihydro-AVAs (DH-AVAs), by both human and mouse S9 fractions. DH-AVAs were detected in the colon and the distal regions but not in the proximal and middle regions of the perfused mouse intestine, and were in specific pathogen-free (SPF) mice but not in GF mice. A kinetic study of humans fed oat bran showed that DH-AVAs reached their maximal concentrations at much later time points than their corresponding AVAs (10.0-15.0 hours vs. 4.0-4.5 hours, respectively). We observed interindividual variations in the metabolism of AVAs to DH-AVAs in humans. Faecalibacterium prausnitzii was identified as the individual bacterium to metabolize AVAs to DH-AVAs by 16S rRNA sequencing analysis. Moreover, as opposed to GF mice, F. prausnitzii-monocolonized mice were able to metabolize AVAs to DH-AVAs. CONCLUSIONS: These findings demonstrate that the presence of intestinal F. prausnitzii is indispensable for proper metabolism of AVAs in both humans and mice. We propose that the abundance of F. prausnitzii can be used to subcategorize individuals into AVA metabolizers and nonmetabolizers after WG oat intake. This study was registered at clinicaltrials.gov as NCT04335435.

Crystal structures of anthranilate phosphoribosyltransferase from Saccharomyces cerevisiae.

Anthranilate phosphoribosyltransferase (AnPRT) catalyzes the transfer of the phosphoribosyl group of 5'-phosphoribosyl-1'-pyrophosphate (PRPP) to anthranilate to form phosphoribosyl-anthranilate. Crystal structures of AnPRTs from bacteria and archaea have previously been determined; however, the structure of Saccharomyces cerevisiae AnPRT (ScAnPRT) still remains unsolved. Here, crystal structures of ScAnPRT in the apo form as well as in complex with its substrate PRPP and the substrate analogue 4-fluoroanthranilate (4FA) are presented. These structures demonstrate that ScAnPRT exhibits the conserved structural fold of type III phosphoribosyltransferase enzymes and shares the similar mode of substrate binding found across the AnPRT protein family. In addition, crystal structures of ScAnPRT mutants (ScAnPRTSer121Ala and ScAnPRTGly141Asn) were also determined. These structures suggested that the conserved residue Ser121 is critical for binding PRPP, while Gly141 is dispensable for binding 4FA. In summary, these structures improved the preliminary understanding of the substrate-binding mode of ScAnPRT and laid foundations for future research.

Engineering of Saccharomyces cerevisiae for anthranilate and methyl anthranilate production.

BACKGROUND: Anthranilate is a platform chemical used by the industry in the synthesis of a broad range of high-value products, such as dyes, perfumes and pharmaceutical compounds. Currently anthranilate is produced via chemical synthesis from non-renewable resources. Biological synthesis would allow the use of renewable carbon sources and avoid accumulation of toxic by-products. Microorganisms produce anthranilate as an intermediate in the tryptophan biosynthetic pathway. Several prokaryotic microorganisms have been engineered to overproduce anthranilate but attempts to engineer eukaryotic microorganisms for anthranilate production are scarce. RESULTS: We subjected Saccharomyces cerevisiae, a widely used eukaryotic production host organism, to metabolic engineering for anthranilate production. A single gene knockout was sufficient to trigger anthranilate accumulation both in minimal and SCD media and the titer could be further improved by subsequent genomic alterations. The effects of the modifications on anthranilate production depended heavily on the growth medium used. By growing an engineered strain in SCD medium an anthranilate titer of 567.9 mg l-1 was obtained, which is the highest reported with an eukaryotic microorganism. Furthermore, the anthranilate biosynthetic pathway was extended by expression of anthranilic acid methyltransferase 1 from Medicago truncatula. When cultivated in YPD medium, this pathway extension enabled production of the grape flavor compound methyl anthranilate in S. cerevisiae at 414 mg l-1. CONCLUSIONS: In this study we have engineered metabolism of S. cerevisiae for improved anthranilate production. The resulting strains may serve as a basis for development of efficient production host organisms for anthranilate-derived compounds. In order to demonstrate suitability of the engineered S. cerevisiae strains for production of such compounds, we successfully extended the anthranilate biosynthesis pathway to synthesis of methyl anthranilate.

The role of anthranilic acid in the increase of depressive symptoms and major depressive disorder during treatment for hepatitis C with pegylated interferon-α2a and oral ribavirin.

Background: Tryptophan metabolism via the kynurenine pathway is considered the link between the immune and endocrine systems. Dysregulation of serotonergic transmission can stem from the direct influence of interferon-α on the activity of serotonergic receptors 5-HT1A and 5-HT2A, and from its indirect effect on tryptophan metabolism. Induction of the kynurenine pathway increases the concentration of neurotoxic kynurenine metabolites, and the activity of kynurenine derivatives is linked to the onset of depression. The aim of our study was to evaluate the relationships between depressive symptoms and kynurenine, tryptophan, anthranilic acid and kynurenic acid concentrations, indolamine 2,3-dioxygenase (IDO) activity and tryptophan availability to the brain. Methods: The study followed a prospective longitudinal cohort design. We evaluated 101 patients with chronic hepatitis C who were treated with pegylated interferon-α2a, and 40 controls who were awaiting treatment. We evaluated the relationships between total score on the Montgomery-Åsberg Depression Rating Scale and kynurenine, tryptophan, anthranilic acid and kynurenic acid concentrations, IDO activity and tryptophan availability to the brain. A logistic regression model was adapted for the diagnosis of major depressive disorder at each time point, taking into account changes in parameters of the kynurenine pathway between a given time point and the baseline measurement. Results: Of the treated patients, 44% fulfilled the criteria for major depressive disorder at least once during the 24 weeks of treatment. Anthranilic acid concentrations were significantly increased compared to baseline for all time points except week 2. Tryptophan availability showed a significant decrease (ß = -0.09, p = 0.01) only in week 12 of treatment. Over time, kynurenine, tryptophan and anthranilic acid concentrations, as well as IDO activity and tryptophan availability to the brain, were significantly associated with total score on the Montgomery-Åsberg Depression Rating Scale. A logistic regression model revealed that participants with decreased tryptophan availability to the brain at 12 weeks of treatment and participants with increased anthranilic acid concentrations at week 24 of treatment were at increased risk for diagnosis of major depressive disorder (odds ratios 2.92 and 3.59, respectively). Limitations: This study had an open-label design in a population receiving naturalistic treatment. Conclusion: The present study provides the first direct evidence of the role of anthranilic acid in the pathogenesis of inflammation-induced major depressive disorder during treatment for hepatitis C with pegylated interferon-α2a.