Validation of aminodeoxychorismate synthase and anthranilate synthase as novel targets for bispecific antibiotics inhibiting conserved protein-protein interactions.

Multi-resistant bacteria are a rapidly emerging threat to modern medicine. It is thus essential to identify and validate novel antibacterial targets that promise high robustness against resistance-mediating mutations. This can be achieved by simultaneously targeting several conserved function-determining protein-protein interactions in enzyme complexes from prokaryotic primary metabolism. Here, we selected two evolutionary related glutamine amidotransferase complexes, aminodeoxychorismate synthase and anthranilate synthase, that are required for the biosynthesis of folate and tryptophan in most prokaryotic organisms. Both enzymes rely on the interplay of a glutaminase and a synthase subunit that is conferred by a highly conserved subunit interface. Consequently, inhibiting subunit association in both enzymes by one competing bispecific inhibitor has the potential to suppress bacterial proliferation. We comprehensively verified two conserved interface hot-spot residues as potential inhibitor-binding sites in vitro by demonstrating their crucial role in subunit association and enzymatic activity. For in vivo target validation, we generated genomically modified Escherichia coli strains in which subunit association was disrupted by modifying these central interface residues. The growth of such strains was drastically retarded on liquid and solid minimal medium due to a lack of folate and tryptophan. Remarkably, the bacteriostatic effect was observed even in the presence of heat-inactivated human plasma, demonstrating that accessible host metabolite concentrations do not compensate for the lack of folate and tryptophan within the tested bacterial cells. We conclude that a potential inhibitor targeting both enzyme complexes will be effective against a broad spectrum of pathogens and offer increased resilience against antibiotic resistance. IMPORTANCE: Antibiotics are indispensable for the treatment of bacterial infections in human and veterinary medicine and are thus a major pillar of modern medicine. However, the exposure of bacteria to antibiotics generates an unintentional selective pressure on bacterial assemblies that over time promotes the development or acquisition of resistance mechanisms, allowing pathogens to escape the treatment. In that manner, humanity is in an ever-lasting race with pathogens to come up with new treatment options before resistances emerge. In general, antibiotics with novel modes of action require more complex pathogen adaptations as compared to chemical derivates of existing entities, thus delaying the emergence of resistance. In this contribution, we use modified Escherichia coli strains to validate two novel targets required for folate and tryptophan biosynthesis that can potentially be targeted by one and the same bispecific protein-protein interaction inhibitor and promise increased robustness against bacterial resistances.

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.

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.

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.

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.

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.

Expression of Brassica napus TTG2, a regulator of trichome development, increases plant sensitivity to salt stress by suppressing the expression of auxin biosynthesis genes.

WRKY transcription factors (TFs) are plant specific and play important roles in regulating diverse biological processes. To identify TFs with broad-spectrum effects on various stress responses in Brassica napus, an important oil crop grown across diverse ecological regions worldwide, we functionally characterized Bna.TTG2 genes, which are homologous to the Arabidopsis AtTTG2 (WRKY44) gene. Four Bna.TTG2 genes were capable of rescuing the trichome phenotypes of Arabidopsis ttg2 mutants. Overexpressing one Bna.TTG2 family member, BnaA.TTG2.a.1, remarkably increased trichome numbers in Arabidopsis and B. napus plants. Interestingly, the BnaA.TTG2.a.1-overexpressing plants of both species exhibited increased sensitivity to salt stress. In BnaA.TTG2.a.1-overexpressing Arabidopsis under salt stress, the endogenous indole-3-acetic acid (IAA) content was reduced, and the expression of two auxin biosynthesis genes, TRYPTOPHAN BIOSYNTHESIS 5 (TRP5) and YUCCA2 (YUC2), was downregulated. The results from yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase reporter assays revealed that BnaA.TTG2.a.1 is able to bind to the promoters of TRP5 and YUC2. These data indicated that BnaA.TTG2.a.1 confers salt sensitivity to overexpressing plants by suppressing the expression of IAA synthesis genes and thus lowering IAA levels. Transgenic Arabidopsis plants with an N-terminus-deleted BnaA.TTG2.a.1 no longer showed hypersensitivity to salt stress, suggesting that the N terminus of BnaA.TTG2.a.1 plays a critical role in salt stress responses. Therefore, in addition to its classical function in trichome development, our study reveals a novel role for Bna.TTG2 genes in salt stress responses.

Conversion of aminodeoxychorismate synthase into anthranilate synthase with Janus mutations: mechanism of pyruvate elimination catalyzed by chorismate enzymes.

The central importance of chorismate enzymes in bacteria, fungi, parasites, and plants combined with their absence in mammals makes them attractive targets for antimicrobials and herbicides. Two of these enzymes, anthranilate synthase (AS) and aminodeoxychorismate synthase (ADCS), are structurally and mechanistically similar. The first catalytic step, amination at C2, is common between them, but AS additionally catalyzes pyruvate elimination, aromatizing the aminated intermediate to anthranilate. Despite prior attempts, the conversion of a pyruvate elimination-deficient enzyme into an elimination-proficient one has not been reported. Janus, a bioinformatics method for predicting mutations required to functionally interconvert homologous enzymes, was employed to predict mutations to convert ADCS into AS. A genetic selection on a library of Janus-predicted mutations was performed. Complementation of an AS-deficient strain of Escherichia coli grown on minimal medium led to several ADCS mutants that allow growth in 6 days compared to 2 days for wild-type AS. The purified mutant enzymes catalyze the conversion of chorismate to anthranilate at rates that are ∼50% of the rate of wild-type ADCS-catalyzed conversion of chorismate to aminodeoxychorismate. The residues mutated do not contact the substrate. Molecular dynamics studies suggest that pyruvate elimination is controlled by the conformation of the C2-aminated intermediate. Enzymes that catalyze elimination favor the equatorial conformation, which presents the C2-H to a conserved active site lysine (Lys424) for deprotonation and maximizes stereoelectronic activation. Acid/base catalysis of pyruvate elimination was confirmed in AS and salicylate synthase by showing incorporation of a solvent-derived proton into the pyruvate methyl group and by solvent kinetic isotope effects on pyruvate elimination catalyzed by AS.

BEM46 shows eisosomal localization and association with tryptophan-derived auxin pathway in Neurospora crassa.

BEM46 proteins are evolutionarily conserved, but their functions remain elusive. We reported previously that the BEM46 protein in Neurospora crassa is targeted to the endoplasmic reticulum (ER) and is essential for ascospore germination. In the present study, we established a bem46 knockout strain of N. crassa. This Δbem46 mutant exhibited a level of ascospore germination lower than that of the wild type but much higher than those of the previously characterized bem46-overexpressing and RNA interference (RNAi) lines. Reinvestigation of the RNAi transformants revealed two types of alternatively spliced bem46 mRNA; expression of either type led to a loss of ascospore germination. Our results indicated that the phenotype was not due to bem46 mRNA downregulation or loss but was caused by the alternatively spliced mRNAs and the peptides they encoded. Using the N. crassa ortholog of the eisosomal protein PILA from Aspergillus nidulans, we further demonstrated the colocalization of BEM46 with eisosomes. Employing the yeast two-hybrid system, we identified a single interaction partner: anthranilate synthase component II (encoded by trp-1). This interaction was confirmed in vivo by a split-YFP (yellow fluorescent protein) approach. The Δtrp-1 mutant showed reduced ascospore germination and increased indole production, and we used bioinformatic tools to identify a putative auxin biosynthetic pathway. The genes involved exhibited various levels of transcriptional regulation in the different bem46 transformant and mutant strains. We also investigated the indole production of the strains in different developmental stages. Our findings suggested that the regulation of indole biosynthesis genes was influenced by bem46 overexpression. Furthermore, we uncovered evidence of colocalization of BEM46 with the neutral amino acid transporter MTR.

Production of indole alkaloids by metabolic engineering of the tryptophan pathway in rice.

Tryptophan decarboxylase (TDC) converts tryptophan (Trp) into tryptamine, consequently increasing the metabolic flow of tryptophan derivatives into the production of secondary metabolites such as indole alkaloids. We inserted an expression cassette containing OsTDC, a putative tryptophan decarboxylase gene from rice, into an expression plasmid vector containing OASA1D, the feedback-resistant anthranilate synthase alpha-subunit mutant (OASA1D). Overexpression of OASA1D has been reported to significantly increase Trp levels in rice. The co-expression of OsTDC and OASA1D in rice calli led to almost complete depletion of the Trp pool and a consequent increase in the tryptamine pool. This indicates that TDC inactivity is a contributory factor for the accumulation of Trp in rice transgenics overexpressing OASA1D. Metabolic profiling of the calli expressing OsTDC and OASA1D revealed the accumulation of serotonin and serotonin-derived indole compounds (potentially pharmacoactive ß-carbolines) that have not been reported from rice. Rice calli overexpressing OASA1D:OASA1D is a novel system for the production of significant amounts of pharmacologically useful indole alkaloids in rice.