De novo transcriptome analysis of Justicia adhatoda reveals candidate genes involved in major biosynthetic pathway.

BACKGROUND: Justicia adhatoda is an important medicinal plant traditionally used in the Indian system of medicine and the absence of molecular-level studies in this plant hinders its wide use, hence the study was aimed to analyse the genes involved in its various pathways. METHODS AND RESULTS: The RNA isolated was subjected to Illumina sequencing. De novo assembly was performed using TRINITY software which produced 171,064 transcripts with 55,528 genes and N50 value of 2065 bp, followed by annotation of unigenes against NCBI, KEGG and Gene ontology databases resulted in 105,572 annotated unigenes and 40,288 non-annotated unigenes. A total of 5980 unigenes were mapped to 144 biochemical pathways, including the metabolism and biosynthesis pathways. The pathway analysis revealed the major transcripts involved in the tryptophan biosynthesis with TPM values of 6.0903, 33.6854, 11.527, 1.6959, and 8.1662 for Anthranilate synthase alpha, Anthranilate synthase beta, Arogenate/Prephenate dehydratase, Chorismate synthase and Chorismate mutase, respectively. The qRT-PCR validation of the key enzymes showed up-regulation in mid mature leaf when compared to root and young leaf tissue. A total of 16,154 SSRs were identified from the leaf transcriptome of J. Adhatoda ,which could be helpful in molecular breeding. CONCLUSIONS: The study aimed at identifying transcripts involved in the tryptophan biosynthesis pathway for its medicinal properties, as it acts as a precursor to the acridone alkaloid biosynthesis with major key enzymes and their validation. This is the first study that reports transcriptome assembly and annotation of J. adhatoda plant.

A multi-omics framework reveals strawberry flavor genes and their regulatory elements.

Flavor is essential to consumer preference of foods and is an increasing focus of plant breeding programs. In fruit crops, identifying genes underlying volatile organic compounds has great promise to accelerate flavor improvement, but polyploidy and heterozygosity in many species have slowed progress. Here we use octoploid cultivated strawberry to demonstrate how genomic heterozygosity, transcriptomic intricacy and fruit metabolomic diversity can be treated as strengths and leveraged to uncover fruit flavor genes and their regulatory elements. Multi-omics datasets were generated including an expression quantitative trait loci map with 196 diverse breeding lines, haplotype-phased genomes of a highly-flavored breeding selection, a genome-wide structural variant map using five haplotypes, and volatile genome-wide association study (GWAS) with > 300 individuals. Overlaying regulatory elements, structural variants and GWAS-linked allele-specific expression of numerous genes to variation in volatile compounds important to flavor. In one example, the functional role of anthranilate synthase alpha subunit 1 in methyl anthranilate biosynthesis was supported via fruit transient gene expression assays. These results demonstrate a framework for flavor gene discovery in fruit crops and a pathway to molecular breeding of cultivars with complex and desirable flavor.

Photoswitching of Feedback Inhibition by Tryptophan in Anthranilate Synthase.

The artificial regulation of enzymatic activity by light is an important goal of synthetic biology that can be achieved by the incorporation of light-responsive noncanonical amino acids via genetic code expansion. Here, we apply this concept to anthranilate synthase from Salmonella typhimurium (stTrpE). This enzyme catalyzes the first step of tryptophan biosynthesis, and its activity is feedback-inhibited by the binding of the end-product of the pathway to an allosteric site. To put this feedback inhibition of stTrpE by tryptophan under the control of light, we individually replaced 15 different amino acid residues with the photosensitive noncanonical amino acid o-nitrobenzyl-O-tyrosine (ONBY). ONBY contains a sterically demanding caging group that was meant to cover the allosteric site. Steady-state enzyme kinetics showed that the negative effect of tryptophan on the catalytic activity of the two variants stTrpE-K50ONBY and stTrpE-Y455ONBY was diminished compared to the wild-type enzyme by 1 to 2 orders of magnitude. Upon light-induced decaging of ONBY to the less space-consuming tyrosine residue, tryptophan binding to the allosteric site was restored and catalytic activity was inhibited almost as efficiently as observed for wild-type stTrpE. Based on these results, direct photocontrol of feedback inhibition of stTrpE-K50ONBY and stTrpE-Y455ONBY could be achieved by irradiation during the reaction. Molecular modeling studies allowed us to rationalize the observed functional conversion from the noninhibited caged to the tryptophan-inhibited decaged states. Our study shows that feedback inhibition, which is an important mechanism to regulate key metabolic enzymes, can be efficiently controlled by the purposeful use of light-responsive noncanonical amino acids.

Azotobacter chroococcum and Pseudomonas putida enhance pyrroloquinazoline alkaloids accumulation in Adhatoda vasica hairy roots by biotization.

Adhatoda vasica is used in the treatment of cold, cough, chronic bronchitis, asthma, diarrhea, and dysentery. The biological activities of this species are attributed with the presence of alkaloids, triterpenoids, and flavonoids. Agrobacterium rhizogenes-mediated transformation of A. vasica, produces pyrroloquinazoline alkaloids, was achieved by infecting leaf discs with strain ATCC15834. The bacterial strain infected 82.7% leaf discs and 5-7 hairy root initials were developed from the cut edges of leaf discs. In this study, seven strains of Azotobacter chroococcum and five strains of Pseudomonas putida were used for the biotization of hairy roots. Plant growth-promoting rhizobacteria (PGPR) develops symbiotic association with roots of plants and increases the growth parameters of plants. PGPR (A. chroococcum and P. putida) increased the profiles of nitrogenase and acid phosphatase enzymes, biomass, dry matter contents, anthranilate synthase activity and accumulation of pyrroloquizoline alkaloids in the biotized hairy roots. Both enzymes (nitrogenase and acid phosphatase) maintain sufficient supply of nitrogen and dissolved phosphorus to the cells of hairy roots therefore, the levels of anthranilate synthase activity and pyrroloquinazoline alkaloids are increased. Total seven pyrroloquinazoline alkaloids (vasicine, vasicinone, vasicine acetate, 2-acetyl benzyl amine, vasicinolone, deoxyvasicine and vasicol) were identified from the biotized hairy roots of A. vasica. In our study, biotization increased the profiles of pyrroloquinazoline alkaloids therefore, this strategy may be used in increasing the production of medicinally important secondary metabolites in other plant species also. Our hypothetical model demonstrates that P. putida cell surface receptors receive root exudates by attaching on hairy roots. After attachment, the bacterial strain penetrates in the biotized hairy roots. This endophytic interaction stimulates acid phosphatase activity in the cells of biotized hairy roots. The P. putida plasmid gene (ppp1) expression led to the synthesis of acid phosphatase in cytosol. The enzyme enhances phosphorus availability as well as induces the formation of phosphoribosyl diphosphate. Later, phosphoribosyl diphosphate metabolizes to tryptophan and finally tryptophan converts to anthranilic acid. The synthesized anthranilic acid used in the synthesis of alkaloids in A. vasica.

Coordinated cytokinin signaling and auxin biosynthesis mediates arsenate-induced root growth inhibition.

Interactions between plant hormones and environmental signals are important for the maintenance of root growth plasticity under ever-changing environmental conditions. Here, we demonstrate that arsenate (AsV), the most prevalent form of arsenic (As) in nature, restrains elongation of the primary root through transcriptional regulation of local auxin biosynthesis genes in the root tips of Arabidopsis (Arabidopsis thaliana) plants. The ANTHRANILATE SYNTHASE ALPHA SUBUNIT 1 (ASA1) and BETA SUBUNIT 1 (ASB1) genes encode enzymes that catalyze the conversion of chorismate to anthranilate (ANT) via the tryptophan-dependent auxin biosynthesis pathway. Our results showed that AsV upregulates ASA1 and ASB1 expression in root tips, and ASA1- and ASB1-mediated auxin biosynthesis is involved in AsV-induced root growth inhibition. Further investigation confirmed that AsV activates cytokinin signaling by stabilizing the type-B ARABIDOPSIS RESPONSE REGULATOR1 (ARR1) protein, which directly promotes the transcription of ASA1 and ASB1 genes by binding to their promoters. Genetic analysis revealed that ASA1 and ASB1 are epistatic to ARR1 in the AsV-induced inhibition of primary root elongation. Overall, the results of this study illustrate a molecular framework that explains AsV-induced root growth inhibition via crosstalk between two major plant growth regulators, auxin and cytokinin.

Reprogramming the Specificity of a Protein Interface by Computational and Data-Driven Design.

The formation of specific protein complexes in a cell is a non-trivial problem given the co-existence of thousands of different polypeptide chains. A particularly difficult case are two glutamine amidotransferase complexes (anthranilate synthase [AS] and aminodeoxychorismate synthase [ADCS]), which are composed of homologous pairs of synthase and glutaminase subunits. We have attempted to identify discriminating interface residues of the glutaminase subunit TrpG from AS, which are responsible for its specific interaction with the synthase subunit TrpEx and prevent binding to the closely related synthase subunit PabB from ADCS. For this purpose, TrpG-specific interface residues were grafted into the glutaminase subunit PabA from ADCS by two different approaches, namely a computational and a data-driven one. Both approaches resulted in PabA variants that bound TrpEx with higher affinity than PabB. Hence, we have accomplished a reprogramming of protein-protein interaction specificity that provides insights into the evolutionary adaptation of protein interfaces.

Structure of subunit I of the anthranilate synthase complex of Mycolicibacterium smegmatis.

The tryptophan biosynthesis pathway, which does not exist in mammals, is highly conserved in Mycobacterium. Anthranilate synthase (AS) catalyzes the initial reactions in the tryptophan biosynthesis pathway in many microorganisms, catalyzing the conversion of glutamine and chorismate to form pyruvate and anthranilate. Here, the crystal structure of anthranilate synthase component I (AS I) from Mycolicibacterium smegmatis (MsTrpE) has been determined to 1.7 Å resolution. MsTrpE crystallizes in the space group P1 with two monomers in the asymmetric unit, which is consistent with the oligomeric state in solution as confirmed by analytical ultracentrifugation. Inspection of the active site shows that it is in the active form with a bound Mg2+ ion and a ligand that is modelled as benzoate. The position of benzoate mimics the position of the anthranilate product in the active site. The structure of MsTrpE will provide a starting point for the investigation of latent biotechnology and pharmaceutical applications of anthranilate synthase component I.

Synthesis, bioactivity, and enzymatic modification of antibacterial thiotetromycin derivatives.

Thiotetronate-containing natural products, including thiolactomycin, thiotetromycin, and thiotetroamide, are potent, broad-spectrum antibacterial compounds that target fatty acid synthesis in bacteria. Natural modifications at the C-5 dialkyl position in this molecular series result in pronounced bioactivity differences. The C-5 acetamide-containing thiotetroamide, which is the more potent antibacterial agent in this family, is biosynthesized from the C-5 ethyl analogue thiotetromycin via a unique two-enzyme process involving the cytochrome P450-amidotransferase enzyme pair TtmP-TtmN. Herein we synthesized a focused library of 17 novel thiotetromycin derivatives differing at the 5-position alkyl substituent to investigate their biological activities and their reactivity towards the hydroxylase TtmP. Although we observed marginal anti-tuberculosis activity, select thiotetromycin analogues showed antibacterial activity against an Escherichia coli ΔtolC strain with IC50 values in a range of 1.9-36 µg mL-1. Additional screening efforts highlighted select thiotetronate analogues as inhibitors of the cancer-associated enzyme nicotinamide N-methyltransferase (NNMT), with a unique scaffold compared to previously identified NNMT inhibitors. In vitro assays further showed that the TtmP P450 was capable of resolving racemic substrate mixtures and had modest promiscuity to hydroxylate derivatives with variable alkyl chains; however triple oxidation to a carboxylic acid remained specific for the natural thiotetromycin substrate. The tendency of TtmP to accept a range of unnatural substrates for hydroxylation makes it an interesting target for P450 engineering towards broader applications.

Gut Microbiota Metabolite Indole Propionic Acid Targets Tryptophan Biosynthesis in Mycobacterium tuberculosis.

Indole propionic acid (IPA), produced by the gut microbiota, is active against Mycobacterium tuberculosisin vitro and in vivo However, its mechanism of action is unknown. IPA is the deamination product of tryptophan (Trp) and thus a close structural analog of this essential aromatic amino acid. De novo Trp biosynthesis in M. tuberculosis is regulated through feedback inhibition: Trp acts as an allosteric inhibitor of anthranilate synthase TrpE, which catalyzes the first committed step in the Trp biosynthesis pathway. Hence, we hypothesized that IPA may mimic Trp as an allosteric inhibitor of TrpE and exert its antimicrobial effect by blocking synthesis of Trp at the TrpE catalytic step. To test our hypothesis, we carried out metabolic, chemical rescue, genetic, and biochemical analyses. Treatment of mycobacteria with IPA inhibited growth and reduced the intracellular level of Trp, an effect abrogated upon supplementation of Trp in the medium. Missense mutations at the allosteric Trp binding site of TrpE eliminated Trp inhibition and caused IPA resistance. In conclusion, we have shown that IPA blocks Trp biosynthesis in M. tuberculosis via inhibition of TrpE by mimicking the physiological allosteric inhibitor of this enzyme.IMPORTANCE New drugs against tuberculosis are urgently needed. The tryptophan (Trp) analog indole propionic acid (IPA) is the first antitubercular metabolite produced by human gut bacteria. Here, we show that this antibiotic blocks Trp synthesis, an in vivo essential biosynthetic pathway in M. tuberculosis Intriguingly, IPA acts by decoupling a bacterial feedback regulatory mechanism: it mimics Trp as allosteric inhibitor of anthranilate synthase, thereby switching off Trp synthesis regardless of intracellular Trp levels. The identification of IPA's target paves the way for the discovery of more potent TrpE ligands employing rational, target-based lead optimization.

First insights of peptidoglycan amidation in Gram-positive bacteria - the high-resolution crystal structure of Staphylococcus aureus glutamine amidotransferase GatD.

Gram-positive bacteria homeostasis and antibiotic resistance mechanisms are dependent on the intricate architecture of the cell wall, where amidated peptidoglycan plays an important role. The amidation reaction is carried out by the bi-enzymatic complex MurT-GatD, for which biochemical and structural information is very scarce. In this work, we report the first crystal structure of the glutamine amidotransferase member of this complex, GatD from Staphylococcus aureus, at 1.85 Å resolution. A glutamine molecule is found close to the active site funnel, hydrogen-bonded to the conserved R128. In vitro functional studies using 1H-NMR spectroscopy showed that S. aureus MurT-GatD complex has glutaminase activity even in the absence of lipid II, the MurT substrate. In addition, we produced R128A, C94A and H189A mutants, which were totally inactive for glutamine deamidation, revealing their essential role in substrate sequestration and catalytic reaction. GatD from S. aureus and other pathogenic bacteria share high identity to enzymes involved in cobalamin biosynthesis, which can be grouped in a new sub-family of glutamine amidotransferases. Given the ubiquitous presence of GatD, these results provide significant insights into the molecular basis of the so far undisclosed amidation mechanism, contributing to the development of alternative therapeutics to fight infections.

Effect of elicitors on the production of pyrroloquinazoline alkaloids by stimulating anthranilate synthase activity in Adhatoda vasica Nees cell cultures.

MAIN CONCLUSION: Pyrroloquinazoline alkaloids are medicinally important compounds, determined by HPLC from cell cultures of Adhatoda vasica . The maximum production of vasicinone (12-fold) and vasicine (8.3-fold) was enhanced by stimulating the anthranilate synthase activity via feeding of tryptophan and sorbitol. The decoction of Adhatoda vasica leaves is used for the treatment of throat irritations, inflammations and recommended as expectorant. The plant species contains pyrroloquinazoline alkaloids and has been reported to demonstrate various biological activities. To investigate the effect of elicitors to increase the production of alkaloids, five groups (auxins and cytokinins, biotic elicitors, polysaccharides, amino acids and salts) of elicitors were evaluated. Maximum production of vasicinone (72.74 ± 0.74 mg/g DW; 12-fold) and vasicine (99.44 ± 0.28 mg/g DW; 8.3-fold) was enhanced by feeding of tryptophan and sorbitol at 50 mM concentration in cell cultures. Fourteen free amino acids were estimated from the elicited cells. Sorbitol stimulated up to a maximum accumulation of serine (8.2-fold). The maximal anthranilate synthase (AS) activity (7.5 ± 0.47 pkat/mg protein; 2.9-fold) was induced by salicylic acid and sorbitol. Anthranilate synthase functions as rate-limiting factor for the biosynthesis of pyrroloquinazoline alkaloids. Our results support the widespread use of tryptophan and sorbitol as elicitors to raise the production of vasicinone, vasicine, 2-acetyl benzyl amine and other pyrroloquinazoline alkaloids in cell cultures of A. vasica.

Indole-3-butyric acid promotes adventitious rooting in Arabidopsis thaliana thin cell layers by conversion into indole-3-acetic acid and stimulation of anthranilate synthase activity.

BACKGROUND: Indole-3-acetic acid (IAA), and its precursor indole-3-butyric acid (IBA), control adventitious root (AR) formation in planta. Adventitious roots are also crucial for propagation via cuttings. However, IBA role(s) is/are still far to be elucidated. In Arabidopsis thaliana stem cuttings, 10 µM IBA is more AR-inductive than 10 µM IAA, and, in thin cell layers (TCLs), IBA induces ARs when combined with 0.1 µM kinetin (Kin). It is unknown whether arabidopsis TCLs produce ARs under IBA alone (10 µM) or IAA alone (10 µM), and whether they contain endogenous IAA/IBA at culture onset, possibly interfering with the exogenous IBA/IAA input. Moreover, it is unknown whether an IBA-to-IAA conversion is active in TCLs, and positively affects AR formation, possibly through the activity of the nitric oxide (NO) deriving from the conversion process. RESULTS: Revealed undetectable levels of both auxins at culture onset, showing that arabidopsis TCLs were optimal for investigating AR-formation under the total control of exogenous auxins. The AR-response of TCLs from various ecotypes, transgenic lines and knockout mutants was analyzed under different treatments. It was shown that ARs are better induced by IBA than IAA and IBA + Kin. IBA induced IAA-efflux (PIN1) and IAA-influx (AUX1/LAX3) genes, IAA-influx carriers activities, and expression of ANTHRANILATE SYNTHASE -alpha1 (ASA1), a gene involved in IAA-biosynthesis. ASA1 and ANTHRANILATE SYNTHASE -beta1 (ASB1), the other subunit of the same enzyme, positively affected AR-formation in the presence of exogenous IBA, because the AR-response in the TCLs of their mutant wei2wei7 was highly reduced. The AR-response of IBA-treated TCLs from ech2ibr10 mutant, blocked into IBA-to-IAA-conversion, was also strongly reduced. Nitric oxide, an IAA downstream signal and a by-product of IBA-to-IAA conversion, was early detected in IAA- and IBA-treated TCLs, but at higher levels in the latter explants. CONCLUSIONS: Altogether, results showed that IBA induced AR-formation by conversion into IAA involving NO activity, and by a positive action on IAA-transport and ASA1/ASB1-mediated IAA-biosynthesis. Results are important for applications aimed to overcome rooting recalcitrance in species of economic value, but mainly for helping to understand IBA involvement in the natural process of adventitious rooting.

Recent developments in the isolation, biological function, biosynthesis, and synthesis of phenazine natural products.

Phenazines are natural products which are produced by bacteria or by archaeal Methanosarcina species. The tricyclic ring system enables redox processes, which producing organisms use for oxidation of NADH or for the generation of reactive oxygen species (ROS), giving them advantages over other microorganisms. In this review we summarize the progress in the field since 2005 regarding the isolation of new phenazine natural products, new insights in their biological function, and particularly the now almost completely understood biosynthesis. The review is complemented by a description of new synthetic methods and total syntheses of phenazines.

Still stable after 11 years: A Catharanthus roseus Hairy root line maintains inducible expression of anthranilate synthase.

Hairy root cultures generated using Agrobacterium rhizogenes are an extensively investigated system for the overproduction of various secondary metabolite based pharmaceuticals and chemicals. This study demonstrated a transgenic Catharanthus roseus hairy root line carrying a feedback-insensitive anthranilate synthase (AS) maintained chemical and genetic stability for 11 years. The AS gene was originally inserted in the hairy root genome under the control of a glucocorticoid inducible promoter. After 11 years continuous maintenance of this hairy root line, genomic PCR of the ASA gene showed the presence of ASA gene in the genome. The mRNA level of AS was induced to 52-fold after feeding the inducer as compared to the uninduced control. The AS enzyme activity was 18.4 nmol/(min*mg) in the induced roots as compared to 2.1 nmol/(min*mg) in the control. In addition, the changes in terpenoid indole alkaloid concentrations after overexpressing AS were tracked over 11 years. The major alkaloid levels in induced and control roots at 11 years are comparable with the metabolite levels at 5 years. This study demonstrates the long term genetic and biochemical stability of hairy root lines, which has important implications for industrial scale applications. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:66-69, 2017.

Uncoupling of an ammonia channel as a mechanism of allosteric inhibition in anthranilate synthase of Serratia marcescens: dynamic and graph theoretical analysis.

Anthranilate synthase (AS) is the first branch node enzyme that catalyzes the conversion of chorismate to anthranilate in the high energy-consuming tryptophan biosynthetic pathway in Serratia marcescens. AS, with an allosterically-bound inhibitor (tryptophan), shows complete inhibition in its catalytic function, but the inhibitor-bound structure is very similar to that of the substrate-bound AS. Even though the reaction mechanisms of several chorismate-utilizing enzymes are known, the unusual structure-function relationship in catalysis and allosteric inhibition of AS by tryptophan, with an insignificant change in structure, remains elusive. In the absence of structural variation, we use an integrated computational approach of coarse-grained protein contact networks, Gaussian network model, and atomistic Molecular Dynamics simulations of the substrate-bound and inhibitor-bound AS structures, and show the role of small but critical allosteric changes that induce complete inhibition of AS activity. We predict, through dynamic correlation studies, perturbation in crucial inter-subunit interactions between the two substrate-binding sites ("ammonia channel") and the allosteric inhibitor-binding site, and identify, through shortest path analysis, the non-active site residues participating in the communication pathways. We argue that such a regulatory mechanism (change in function without a significant change in the structure) for catalysis is useful for a branch point enzyme that has to undergo fast redistribution of fluxes according to different metabolic states of the organism. Being essential to the survival of microorganisms, including pathogenic ones, and absent in mammals, AS is a highly attractive drug target. Thus, the allosteric AS residues participating in catalysis identified in this study could be important for drugability.

Examining the transcriptional response of overexpressing anthranilate synthase in the hairy roots of an important medicinal plant Catharanthus roseus by RNA-seq.

BACKGROUND: Clinically important anti-cancer drugs vinblastine and vincristine are solely synthesized by the terpenoid indole alkaloid (TIA) pathway in Catharanthus roseus. Anthranilate synthase (AS) is a rate-limiting enzyme in the TIA pathway. The transgenic C. roseus hairy root line overexpressing a feedback insensitive ASα subunit under the control of an inducible promoter and the ASß subunit constitutively was previously created for the overproduction of TIAs. However, both increases and decreases in TIAs were detected after overexpressing ASα. Although genetic modification is targeted to one gene in the TIA pathway, it could trigger global transcriptional changes that can directly or indirectly affect TIA biosynthesis. In this study, Illumina sequencing and RT-qPCR were used to detect the transcriptional responses to overexpressing AS, which can increase understanding of the complex regulation of the TIA pathway and further inspire rational metabolic engineering for enhanced TIA production in C. roseus hairy roots. RESULTS: Overexpressing AS in C. roseus hairy roots altered the transcription of most known TIA pathway genes and regulators after 12, 24, and 48 h induction detected by RT-qPCR. Changes in the transcriptome of C. roseus hairy roots was further investigated 18 hours after ASα induction and compared to the control hairy roots using RNA-seq. A unigene set of 30,281 was obtained by de novo assembly of the sequencing reads. Comparison of the differentially expressed transcriptional profiles resulted in 2853 differentially expressed transcripts. Functional annotation of these transcripts revealed a complex and systematically transcriptome change in ASαß hairy roots. Pathway analysis shows alterations in many pathways such as aromatic amino acid biosynthesis, jasmonic acid (JA) biosynthesis and other secondary metabolic pathways after perturbing AS. Moreover, many genes in overall stress response were differentially expressed after overexpressing ASα. CONCLUSION: The transcriptomic analysis illustrates overexpressing AS stimulates the overall stress response and affects the metabolic networks in C. roseus hairy roots. The up-regulation of endogenous JA biosynthesis pathway indicates the involvement of JA signal transduction to regulate TIA biosynthesis in ASαß engineered roots and explained why many of the transcripts for TIA genes and regulators are seen to increase with AS overexpression.

An Unexpected Route to an Essential Cofactor: Escherichia coli Relies on Threonine for Thiamine Biosynthesis.

UNLABELLED: Metabolism consists of biochemical reactions that are combined to generate a robust metabolic network that can respond to perturbations and also adapt to changing environmental conditions. Escherichia coli and Salmonella enterica are closely related enterobacteria that share metabolic components, pathway structures, and regulatory strategies. The synthesis of thiamine in S. enterica has been used to define a node of the metabolic network by analyzing alternative inputs to thiamine synthesis from diverse metabolic pathways. To assess the conservation of metabolic networks in organisms with highly conserved components, metabolic contributions to thiamine synthesis in E. coli were investigated. Unexpectedly, we found that, unlike S. enterica, E. coli does not use the phosphoribosylpyrophosphate (PRPP) amidotransferase (PurF) as the primary enzyme for synthesis of phosphoribosylamine (PRA). In fact, our data showed that up to 50% of the PRA used by E. coli to make thiamine requires the activities of threonine dehydratase (IlvA) and anthranilate synthase component II (TrpD). Significantly, the IlvA- and TrpD-dependent pathway to PRA functions in S. enterica only in the absence of a functional reactive intermediate deaminase (RidA) enzyme, bringing into focus how these closely related bacteria have distinct metabolic networks. IMPORTANCE: In most bacteria, including Salmonella strains and Escherichia coli, synthesis of the pyrimidine moiety of the essential coenzyme, thiamine pyrophosphate (TPP), shares enzymes with the purine biosynthetic pathway. Phosphoribosylpyrophosphate amidotransferase, encoded by the purF gene, generates phosphoribosylamine (PRA) and is considered the first enzyme in the biosynthesis of purines and the pyrimidine moiety of TPP. We show here that, unlike Salmonella, E. coli synthesizes significant thiamine from PRA derived from threonine using enzymes from the isoleucine and tryptophan biosynthetic pathways. These data show that two closely related organisms can have distinct metabolic network structures despite having similar enzyme components, thus emphasizing caveats associated with predicting metabolic potential from genome content.

Arabidopsis ERF1 Mediates Cross-Talk between Ethylene and Auxin Biosynthesis during Primary Root Elongation by Regulating ASA1 Expression.

The gaseous phytohormone ethylene participates in the regulation of root growth and development in Arabidopsis. It is known that root growth inhibition by ethylene involves auxin, which is partially mediated by the action of the WEAK ETHYLENE INSENSITIVE2/ANTHRANILATE SYNTHASE α1 (WEI2/ASA1), encoding a rate-limiting enzyme in tryptophan (Trp) biosynthesis, from which auxin is derived. However, the molecular mechanism by which ethylene decreases root growth via ASA1 is not understood. Here we report that the ethylene-responsive AP2 transcription factor, ETHYLENE RESPONSE FACTOR1 (ERF1), plays an important role in primary root elongation of Arabidopsis. Using loss- and gain-of-function transgenic lines as well as biochemical analysis, we demonstrate that ERF1 can directly up-regulate ASA1 by binding to its promoter, leading to auxin accumulation and ethylene-induced inhibition of root growth. This discloses one mechanism linking ethylene signaling and auxin biosynthesis in Arabidopsis roots.

TrpE feedback mutants reveal roadblocks and conduits toward increasing secondary metabolism in Aspergillus fumigatus.

Small peptides formed from non-ribosomal peptide synthetases (NRPS) are bioactive molecules produced by many fungi including the genus Aspergillus. A subset of NRPS utilizes tryptophan and its precursor, the non-proteinogenic amino acid anthranilate, in synthesis of various metabolites such as Aspergillus fumigatus fumiquinazolines (Fqs) produced by the fmq gene cluster. The A. fumigatus genome contains two putative anthranilate synthases - a key enzyme in conversion of anthranilic acid to tryptophan - one beside the fmq cluster and one in a region of co-linearity with other Aspergillus spp. Only the gene found in the co-linear region, trpE, was involved in tryptophan biosynthesis. We found that site-specific mutations of the TrpE feedback domain resulted in significantly increased production of anthranilate, tryptophan, p-aminobenzoate and fumiquinazolines FqF and FqC. Supplementation with tryptophan restored metabolism to near wild type levels in the feedback mutants and suggested that synthesis of the tryptophan degradation product kynurenine could negatively impact Fq synthesis. The second putative anthranilate synthase gene next to the fmq cluster was termed icsA for its considerable identity to isochorismate synthases in bacteria. Although icsA had no impact on A. fumigatus Fq production, deletion and over-expression of icsA increased and decreased respectively aromatic amino acid levels suggesting that IcsA can draw from the cellular chorismate pool.

Structure and inhibition of subunit I of the anthranilate synthase complex of Mycobacterium tuberculosis and expression of the active complex.

The tryptophan-biosynthesis pathway is essential for Mycobacterium tuberculosis (Mtb) to cause disease, but not all of the enzymes that catalyse this pathway in this organism have been identified. The structure and function of the enzyme complex that catalyses the first committed step in the pathway, the anthranilate synthase (AS) complex, have been analysed. It is shown that the open reading frames Rv1609 (trpE) and Rv0013 (trpG) encode the chorismate-utilizing (AS-I) and glutamine amidotransferase (AS-II) subunits of the AS complex, respectively. Biochemical assays show that when these subunits are co-expressed a bifunctional AS complex is obtained. Crystallization trials on Mtb-AS unexpectedly gave crystals containing only AS-I, presumably owing to its selective crystallization from solutions containing a mixture of the AS complex and free AS-I. The three-dimensional structure reveals that Mtb-AS-I dimerizes via an interface that has not previously been seen in AS complexes. As is the case in other bacteria, it is demonstrated that Mtb-AS shows cooperative allosteric inhibition by tryptophan, which can be rationalized based on interactions at this interface. Comparative inhibition studies on Mtb-AS-I and related enzymes highlight the potential for single inhibitory compounds to target multiple chorismate-utilizing enzymes for TB drug discovery.