Aminodeoxychorismate Synthase Research Articles

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

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.

Structural basis for the allosteric pathway of 4-amino-4-deoxychorismate synthase.

4-Amino-4-deoxychorismate synthase (ADCS), a chorismate-utilizing enzyme, is composed of two subunits: PabA and PabB. PabA is a glutamine amidotransferase that hydrolyzes glutamine into glutamate and ammonia. PabB is an aminodeoxychorismate synthase that converts chorismate to 4-amino-4-deoxychorismate (ADC) using the ammonia produced by PabA. ADCS functions under allosteric regulation between PabA and PabB. However, the allosteric mechanism remains unresolved because the structure of the PabA-PabB complex has not been determined. Here, the crystal structure and characterization of PapA from Streptomyces venezuelae (SvPapA), a bifunctional enzyme comprising the PabA and PabB domains, is reported. SvPapA forms a unique dimer in which PabA and PabB domains from different monomers complement each other and form an active structure. The chorismate-bound structure revealed that recognition of the C1 carboxyl group by Thr501 and Gly502 of the 498-PIKTG-502 motif in the PabB domain is essential for the catalytic Lys500 to reach the C2 atom, a reaction-initiation site. SvPapA demonstrated ADCS activity in the presence of Mg2+ when glutamate or NH+4 was used as the amino donor. The crystal structure indicated that the Mg2+-binding position changed depending on the binding of chorismate. In addition, significant structural changes were observed in the PabA domain depending on the presence or absence of chorismate. This study provides insights into the structural factors that are involved in the allosteric regulation of ADCS.

Redefining genomic view of Clostridioides difficile through pangenome analysis and identification of drug targets from its core genome.

Introduction: Clostridioides difficile infection (CDI) is a leading cause of gastrointestinal infections and in the present day is a major concern for global health care system. The unavailability of specific antibiotics for CDI treatment and its emerging cases worldwide further broaden the challenge to control CDI. Methods: The availability of a large number of genome sequences for C. difficile and many bioinformatics tools for genome analysis provides the opportunity for in silico pangenomic analysis. In the present study, 97 strains of C. difficilewere used for pangenomic studies and characterized for their phylogenomic and functional analysis. Results: Pangenome analysis reveals open pangenome of C. difficile and high genetic diversity. Sequence and interactome analysis of 1,481 core genes was done and eight potent drug targets are identified. Three drug targets, namely, aminodeoxychorismate synthase (PabB), d-alanyl-d-alanine carboxypeptidase (DD-CPase) and undecaprenyl diphospho-muramoyl pentapeptide beta-N-acetylglucosaminyl transferase (MurG transferase), have been reported as drug targets for other human pathogens, and five targets, namely, bifunctional diguanylate cyclase/phosphodiesterase (cyclic-diGMP), sporulation transcription factor (Spo0A), histidinol-phosphate transaminase (HisC), 3-deoxy-7-phosphoheptulonate synthase (DAHP synthase) and c-di-GMP phosphodiesterase (PdcA), are novel. Conclusion: The suggested potent targets could act as broad-spectrum drug targets for C. difficile. However, further validation needs to be done before using them for lead compound discovery.

Discovery of brevijanazines from Aspergillus brevijanus reveals the molecular basis for p-nitrobenzoic acid in fungi.

The brevijanazines are novel p-nitrobenzoylated piperazines isolated from Aspergillus brevijanus. Their structures were elucidated by spectroscopic analysis, X-ray crystallography and total synthesis. Heterologous biosynthesis, precursor feeding and in vitro microsomal assays unveiled the biosynthetic pathway to the brevijanazines, featuring a cytochrome P450 oxygenase that converts p-aminobenzoic acid to p-nitrobenzoic acid.

Red light enhances folate accumulation in wheat seedlings.

Red, white, blue, green, and yellow lights were applied to investigate their effects on folate accumulation in wheat seedlings. The different lights, especially red light, significantly increased the total folate content. Total folate showed maximum accumulation under 30 μmol/(m2·s) of red light, with an increase of 24% compared with the control (darkness). 5-Methyl-tetrahydrofolate (5-CH3-THF) was the dominant folate component, and was significantly increased by red light irradiation. In addition, under red light, the folate content of leaves was higher and more sensitive to light than that of endosperm or roots. Red light up-regulated the expression of guanosine triphosphate (GTP) cyclohydrolase 1 (GCH1) and aminodeoxychorismate synthase(ADCS), enhanced the activity of GCH1 and ADCS, and increased the content of precursors of folate synthesis, including pterin and p-aminobenzoic acid (pABA). Hence, the increased folate accumulation promoted by light could be attributed to the increased content of folate synthesis precursors, the activity of key enzymes, and related gene expression.

Mutations in the Physcomitrium patens gene encoding Aminodeoxychorismate Synthase confer auxotrophic phenotypes.

To facilitate genetic mapping of developmental mutants of Physcomitrium patens, we produced a genetic marker that combines recessive auxotrophy with dominant positive selection. We first identified the gene affected by the pabB4 auxotrophic mutation and then replaced it with a cassette that confers antibiotic resistance. This strain may be used to produce bi-parental somatic hybrids with nearly any other strain.

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.

Genetic and physiological regulation of folate in pak choi (Brassica rapa subsp. Chinensis) germplasm.

Folates are one of the essential micronutrients for all living organisms. Due to inadequate dietary intake, folate deficiency remains prevalent in humans. Genetically diverse germplasms can potentially be used as parents in breeding programs and also for understanding the folate regulatory network. Therefore, we investigated the natural genetic diversity of folates and their physiological regulation in pak choi (Brassica rapa subsp. Chinensis) germplasm. The total folate concentration ranged from 52.7 μg 100 gFW-1 to 166.9 μg 100 gFW-1, with 3.2-fold variation. The main folate vitamer was represented by 5-CH3-H4folate, with 4.5-fold variation. The activities of GTP cyclohydrolase I and aminodeoxy chorismate synthase, the first step of folate synthesis, were high in high folate accessions and low in low folate accessions. Analysis of the transcription levels of 11 genes associated with folate metabolism demonstrated that the difference in folate concentrations may be primarily controlled at the post-transcriptional level. A general correlation between total folate and their precursors was observed. Folate diversity and chlorophyll content were tightly regulated through the methyl cycle. The diverse genetic variation in pak choi germplasm indicated the great genetic potential to integrate breeding programs for folate biofortification and unravel the physiological basis of folate homeostasis in planta.

Effects of temperature stress on the accumulation of ascorbic acid and folates in sweet corn (Zea mays L.) seedlings.

Extreme temperatures are among the primary abiotic stresses that affect plant growth and development. Ascorbic acid (AsA) is an efficient antioxidant for scavenging relative oxygen species accumulated under stress. Folates play a significant role in DNA synthesis and protect plants against oxidative stress. Sweet corn (Zea mays L.), a crop grown worldwide, is sensitive to extreme temperatures at seedling stage, which may cause yield loss. This study was conducted to explore the biosynthetic regulative mechanism of AsA and folates in sweet corn seedlings under temperature stress. The AsA and folate composition and relative gene expression in sweet corn seedlings grown under different temperature stresses (10, 25, and 40 °C) were evaluated. The imposition of temperature stress altered the AsA content mainly by modulating the expression of Zm DHAR, whose encoded enzyme dehydroascorbic reductase (DHAR) is essential in the AsA recycle pathway. Low temperature stress raised the expressions of relative genes, leading to folate accumulation. High temperature stress modulated the folate content by influencing the expression of the correspondence gene for aminodeoxychorismate synthase, Zm ADCS, as well as downstream genes that connected with DNA methylation. These results provided a theoretical basis, at a genetic level, for understanding the stress responses mechanism in sweet corn seedlings, offering guidance for sweet corn cultivation. © 2019 Society of Chemical Industry.

Phylogeny and Sequence Space: A Combined Approach to Analyze the Evolutionary Trajectories of Homologous Proteins. The Case Study of Aminodeoxychorismate Synthase.

During the course of evolution, variations of a protein sequence is an ongoing phenomenon however limited by the need to maintain its structural and functional integrity. Deciphering the evolutionary path of a protein is thus of fundamental interest. With the development of new methods to visualize high dimension spaces and the improvement of phylogenetic analysis tools, it is possible to study the evolutionary trajectories of proteins in the sequence space. Using the data-driven high-dimensional scaling method, we show that it is possible to predict and represent potential evolutionary trajectories by representing phylogenetic trees into a 3D projection of the sequence space. With the case of the aminodeoxychorismate synthase, an enzyme involved in folate synthesis, we show that this representation raises interesting questions about the complexity of the evolution of a given biological function, in particular concerning its capacity to explore the sequence space.

Mapping the Allosteric Communication Network of Aminodeoxychorismate Synthase

Allosteric communication between different subunits in metabolic enzyme complexes is of utmost physiological importance but only understood for few systems. We analyzed the structural basis of allostery in aminodeoxychorismate synthase (ADCS), which is a member of the family of glutamine amidotransferases and catalyzes the committed step of the folate biosynthetic pathway. ADCS consists of the synthase subunit PabB and the glutaminase subunit PabA, which is allosterically stimulated by the presence of the PabB substrate chorismate. We first solved the crystal structure of a PabA subunit at 1.9-Å resolution. Based on this structure and the known structure of PabB, we computed an atomic model for the ADCS complex. We then used alanine scanning to test the functional role of 59 conserved residues located between the active sites of PabB and PabA. Steady-state kinetic characterization revealed four branches of a conserved network of mainly charged residues that propagate the signal from chorismate at the PabB active site to the PabA active site. The branches eventually lead to activity-inducing transformations at (i) the oxyanion hole motif, (ii) the catalytic Cys‐His‐Glu triad, and (iii) glutamine binding residues at the PabA active site. We compare our findings with previously postulated activation mechanisms of different glutamine amidotransferases and propose a unifying regulation mechanism for this ubiquitous family of enzymes.

Metabolic engineering of Bacillus subtilis for production of para-aminobenzoic acid - unexpected importance of carbon source is an advantage for space application.

SummaryHigh‐strength polymers, such as aramid fibres, are important materials in space technology. To obtain these materials in remote locations, such as Mars, biological production is of interest. The aromatic polymer precursor para‐aminobenzoic acid (pABA) can be derived from the shikimate pathway through metabolic engineering of Bacillus subtilis, an organism suited for space synthetic biology. Our engineering strategy included repair of the defective indole‐3‐glycerol phosphate synthase (trpC), knockout of one chorismate mutase isozyme (aroH) and overexpression of the aminodeoxychorismate synthase (pabAB) and aminodeoxychorismate lyase (pabC) from the bacteria Corynebacterium callunae and Xenorhabdus bovienii respectively. Further, a fusion‐protein enzyme (pabABC) was created for channelling of the carbon flux. Using adaptive evolution, mutants of the production strain, able to metabolize xylose, were created, to explore and compare pABA production capacity from different carbon sources. Rather than the efficiency of the substrate or performance of the biochemical pathway, the product toxicity, which was strongly dependent on the pH, appeared to be the overall limiting factor. The highest titre achieved in shake flasks was 3.22 g l−1 with a carbon yield of 12.4% [C‐mol/C‐mol] from an amino sugar. This promises suitability of the system for in situ resource utilization (ISRU) in space biotechnology, where feedstocks that can be derived from cyanobacterial cell lysate play a role.

Improved folate accumulation in genetically modified maize and wheat.

Folates are indispensable co-factors for one-carbon metabolism in all organisms. In humans, suboptimal folate intake results in serious disorders. One promising strategy for improving human folate status is to enhance folate levels in food crops by metabolic engineering. In this study, we cloned two GmGCHI (GTP cyclohydrolase I) genes (Gm8gGCHI and Gm3gGCHI) and one GmADCS (aminodeoxychorismate synthase) gene from soybean, which are responsible for synthesizing the folate precursors pterin and p-aminobenzoate, respectively. We initially confirmed their functions in transgenic Arabidopsis plants and found that Gm8gGCHI increased pterin and folate production more than Gm3gGCHI did. We then co-expressed Gm8gGCHI and GmADCS driven by endosperm-specific promoters in maize and wheat, two major staple crops, to boost their folate metabolic flux. A 4.2-fold and 2.3-fold increase in folate levels were observed in transgenic maize and wheat grains, respectively. To optimize wheat folate enhancement, codon-optimized Gm8gGCHI and tomato LeADCS genes under the control of a wheat endosperm-specific glutenin promoter (1Dx5) were co-transformed. This yielded a 5.6-fold increase in folate in transgenic wheat grains (Gm8gGCHI+/LeADCS+). This two-gene co-expression strategy therefore has the potential to greatly enhance folate levels in maize and wheat, thus improving their nutritional value.

Plasmodium Para-Aminobenzoate Synthesis and Salvage Resolve Avoidance of Folate Competition and Adaptation to Host Diet

Folate metabolism is essential for DNA synthesis and a validated drug target in fast-growing cell populations, including tumors and malaria parasites. Genome data suggest that Plasmodium has retained its capacity to generate folates de novo. However, the metabolic plasticity of folate uptake and biosynthesis by the malaria parasite remains unresolved. Here, we demonstrate that Plasmodium uses an aminodeoxychorismate synthase and an aminodeoxychorismate lyase to promote the biogenesis of the central folate precursor para-aminobenzoate (pABA) in the cytoplasm. We show that the parasite depends on de novo folate synthesis only when dietary intake of pABA by the mammalian host is restricted and that only pABA, rather than fully formed folate, is taken up efficiently. This adaptation, which readily adjusts infection to highly variable pABA levels in the mammalian diet, is specific to blood stages and may have evolved to avoid folate competition between the parasite and its host.

Molecular characterization of dihydroneopterin aldolase and aminodeoxychorismate synthase in common bean-genes coding for enzymes in the folate synthesis pathway.

Common beans (Phaseolus vulgaris) are excellent sources of dietary folates, but different varieties contain different amounts of these compounds. Genes coding for dihydroneopterin aldolase (DHNA) and aminodeoxychorismate synthase (ADCS) of the folate synthesis pathway were characterized by PCR amplification, BAC clone sequencing, and whole genome sequencing. All DHNA and ADCS genes in the Mesoamerican cultivar OAC Rex were isolated and compared with those genes in the genome of Andean genotype G19833. Both genotypes have two functional DHNA genes and one pseudo gene. PvDHNA1 and PvDHNA2 proteins have similar secondary structures and conserved residues as DHNA homologs in Staphylococcus aureus and Arabidopsis. Sequence analysis and synteny mapping indicated that PvDHNA1 might be a duplicated and transposed copy of PvDHNA2. There is only one ADCS gene (PvADCS) identified in the bean genome and it is identical in OAC Rex and G19833. PvADCS has the conserved motifs required for catalytic activity similar to other plant ADCS homologs. DHNA and ADCS gene-specific markers were developed, mapped, and compared to their physical locations on chromosomes 1 and 7, respectively. The gene-specific markers developed in this study should be useful for detection and selection of varieties with enhanced folate contents in bean breeding programs.

Site-directed mutant libraries for isolating minimal mutations yielding functional changes.

Powerful, facile new ways to create libraries of site-directed mutants are demonstrated. These include: (1) one-pot-PCR, (2) multi-pot-PCR, and (3) split-mix-PCR. One-pot-PCR uses mutant oligonucleotides to generate megaprimers in situ, and it was used to randomly incorporate 28 mutations in a gabT gene in a single reaction. In more difficult cases, multi-pot-PCR can be employed: mutant megaprimers are synthesized individually, then combined in a single mutagenesis PCR. This method was used to incorporate 14 out of 15 mutations in a pabB gene. Split-mix-PCR is a conceptually novel method for creation of site-directed mutant libraries. Separate PCRs for each mutant primer are performed, followed by pooling the products of the individual reactions. The pooled mixture is re-aliquoted into individual mutant oligonucleotide PCRs. These steps are repeated for each cycle. Split-mix-PCR results in a nearly random distribution of mutation sites, and a distribution of number-of-mutations per gene that is computable and narrow. Split-mix-PCR was applied to the directed evolution of aminodeoxychorismate synthase into anthranilate synthase, and easily allowed the determination of the fewest mutations required for introduction of novel activity.

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.

Cloning, expression, and characterization of para-aminobenzoic acid (PABA) synthase from Agaricus bisporus 02, a thermotolerant mushroom strain.

The pabS gene of Agaricus bisporus 02 encoding a putative PABA synthase was cloned, and then the recombinant protein was expressed in Escherichia coli BL21 under the control of the T7 promoter. The enzyme with an N-terminal GST tag or His tag, designated GST-AbADCS or His-AbADCS, was purified with glutathione Sepharose 4B or Ni Sepharose 6 Fast Flow. The enzyme was an aminodeoxychorismate synthase, and it was necessary to add with an aminodeoxychorismate lyase for synthesizing PABA. AbADCS has maximum activity at a temperature of approximately 25°C and pH 8.0. Magnesium or manganese ions were necessary for the enzymatic activity. The Michaelis-Menten constant for chorismate was 0.12 mM, and 2.55 mM for glutamine. H2O2 did distinct damage on the activity of the enzyme, which could be slightly recovered by Hsp20. Sulfydryl reagents could remarkably promote its activity, suggesting that cysteine residues are essential for catalytic function.

Overexpression of folate biosynthesis genes in rice (Oryza sativa L.) and evaluation of their impact on seed folate content.

Folate (vitamin B9) deficiency is a global health problem especially in developing countries where the major staple foods such as rice contain extremely low folates. Biofortification of rice could be an alternative complement way to fight folate deficiency. In this study, we evaluated the availability of the genes in each step of folate biosynthesis pathway for rice folate enhancement in the japonica variety kitaake genetic background. The first enzymes GTP cyclohydrolase I (GTPCHI) and aminodeoxychorismate synthase (ADCS) in the pterin and para-aminobenzoate branches resulted in significant increase in seed folate content, respectively (P < 0.01). Overexpression of two closely related enzymes dihydrofolate synthase (DHFS) and folypolyglutamate synthase (FPGS), which perform the first and further additions of glutamates, produced slightly increase in seed folate content separately. The GTPCHI transgene was combined with each of the other transgenes except ADCS to investigate the effects of gene stacking on seed folate accumulation. Seed folate contents in the gene-stacked plants were higher than the individual low-folate transgenic parents, but lower than the high-folate GTPCHI transgenic lines, pointing to an inadequate supply of para-aminobenzoic acid (PABA) precursor initiated by ADCS in constraining folate overproduction in gene-stacked plants.

New gene responsible for para-aminobenzoate biosynthesis

Folate is an essential cofactor in all living cells for one-carbon transfer reactions. para-Aminobenzoate (pABA), a building block of folate, is usually derived from chorismate in the shikimate pathway by reactions of aminodeoxychorismate synthase (PabA and -B) and 4-amino-4-deoxychorismate lyase (PabC). We previously suggested that an alternative pathway for pABA biosynthesis would operate in some microorganisms such as Lactobacillus fermentum and Nitrosomonas europaea since these bacteria showed a prototrophic phenotype to pABA despite the fact that there are no orthologs of pabA, -B, and -C in their genome databases. In this study, a gene of unknown function, NE1434, was obtained from N.europaea by shotgun cloning using a pABA-auxotrophic Escherichia coli mutant (ΔpabABC) as a host. A tracer experiment using [U-(13)C6]glucose suggested that pABA was de novo synthesized in the transformant. An E.coli ΔpabABCΔaroB mutant carrying the NE1434 gene exhibited a prototrophic phenotype to pABA, suggesting that compounds in the shikimate pathway including chorismate were not utilized as substrates by NE1434. Moreover, the CT610 gene, an ortholog of NE1434 located in the folate biosynthetic gene cluster in Chlamydia trachomatis, also complemented pABA-auxotrophic E.coli mutants. Taken together, these results suggest that NE1434 and CT610 participate in pABA biosynthesis.