Identification and characterization of a pab gene cluster responsible for the 4-aminobenzoate degradation pathway, including its involvement in the formation of a γ-glutamylated intermediate in Paraburkholderia terrae strain KU-15.

Paraburkholderia terrae strain KU-15 grows on 2- and 4-nitrobenzoate and 2- and 4-aminobenzoate (ABA) as the sole nitrogen and carbon sources. The genes responsible for the potential degradation of 2- and 4-nitrobenzoate and 2-ABA have been predicted from its genome sequence. In this study, we identified the pab operon in P. terrae strain KU-15. This operon is responsible for the 4-ABA degradation pathway, which involves the formation of a γ-glutamylated intermediate. Reverse transcription-polymerase chain reaction revealed that the pab operon was induced by 4-ABA. Herein, studying the deletion of pabA and pabB1 in strain KU-15 and the examining of Escherichia coli expressing the pab operon revealed the involvement of the operon in 4-ABA degradation. The first step of the degradation pathway is the formation of a γ-glutamylated intermediate, whereby 4-ABA is converted to γ-glutamyl-4-carboxyanilide (γ-GCA). Subsequently, γ-GCA is oxidized to protocatechuate. Overexpression of various genes in E. coli and purification of recombinant proteins permitted the functional characterization of relevant pathway proteins: PabA is a γ-GCA synthetase, PabB1-B3 functions in a multicomponent dioxygenase system responsible for γ-GCA dioxygenation, and PabC is a γ-GCA hydrolase that reverses the formation of γ-GCA by PabA.

Excision of a Protein-Derived Amine for p-Aminobenzoate Assembly by the Self-Sacrificial Heterobimetallic Protein CADD.

Chlamydia protein associating with death domains (CADD), the founding member of a recently discovered class of nonheme dimetal enzymes termed hemeoxygenase-like dimetaloxidases (HDOs), plays an indispensable role in pathogen survival. CADD orchestrates the biosynthesis of p-aminobenzoic acid (pABA) for integration into folate via the self-sacrificial excision of a protein-derived tyrosine (Tyr27) and several additional processing steps, the nature and timing of which have yet to be fully clarified. Nuclear magnetic resonance (NMR) and proteomics approaches reveal the source and probable timing of amine installation by a neighboring lysine (Lys152). Turnover studies using limiting O2 have identified a para-aminobenzaldehyde (pABCHO) metabolic intermediate that is formed on the path to pABA formation. The use of pABCHO and other probe substrates shows that the heterobimetallic Fe/Mn form of the enzyme is capable of oxygen insertion to generate the pABA-carboxylate.

Self-sacrificial tyrosine cleavage by an Fe:Mn oxygenase for the biosynthesis of para-aminobenzoate in Chlamydia trachomatis.

Chlamydia protein associating with death domains (CADD) is involved in the biosynthesis of para-aminobenzoate (pABA), an essential component of the folate cofactor that is required for the survival and proliferation of the human pathogen Chlamydia trachomatis. The pathway used by Chlamydiae for pABA synthesis differs from the canonical multi-enzyme pathway used by most bacteria that relies on chorismate as a metabolic precursor. Rather, recent work showed pABA formation by CADD derives from l-tyrosine. As a member of the emerging superfamily of heme oxygenase-like diiron oxidases (HDOs), CADD was proposed to use a diiron cofactor for catalysis. However, we report maximal pABA formation by CADD occurs upon the addition of both iron and manganese, which implicates a heterobimetallic Fe:Mn cluster is the catalytically active form. Isotopic labeling experiments and proteomics studies show that CADD generates pABA from a protein-derived tyrosine (Tyr27), a residue that is ∼14 Šfrom the dimetal site. We propose that this self-sacrificial reaction occurs through O2 activation by a probable Fe:Mn cluster through a radical relay mechanism that connects to the "substrate" Tyr, followed by amination and direct oxygen insertion. These results provide the molecular basis for pABA formation in C. trachomatis, which will inform the design of novel therapeutics.

Development of CD133 Targeting Multi-Drug Polymer Micellar Nanoparticles for Glioblastoma - In Vitro Evaluation in Glioblastoma Stem Cells.

PURPOSE: Glioblastoma (GBM) is a malignant brain tumor with a poor long-term prognosis due to recurrence from highly resistant GBM cancer stem cells (CSCs), for which the current standard of treatment with temozolomide (TMZ) alone will unlikely produce a viable cure. In addition, CSCs regenerate rapidly and overexpress methyl transferase which overrides the DNA-alkylating mechanism of TMZ, leading to resistance. The objective of this research was to apply the concepts of nanotechnology to develop a multi-drug therapy, TMZ and idasanutlin (RG7388, a potent mouse double minute 2 (MDM2) antagonist), loaded in functionalized nanoparticles (NPs) that target the GBM CSC subpopulation, reduce the cell viability and provide possibility of in vivo preclinical imaging. METHODS: Polymer-micellar NPs composed of poly(styrene-b-ethylene oxide) (PS-b-PEO) and poly(lactic-co-glycolic) acid (PLGA) were developed by a double emulsion technique loading TMZ and/or RG7388. The NPs were covalently bound to a 15-nucleotide base-pair CD133 aptamer to target the CD133 antigen expressed on the surfaces of GBM CSCs. For diagnostic functionality, the NPs were labelled with radiotracer Zirconium-89 (89Zr). RESULTS: NPs maintained size range less than 100 nm, a low negative charge and exhibited the ability to target and kill the CSC subpopulation when TMZ and RG7388 were used in combination. The targeting function of CD133 aptamer promoted killing in GBM CSCs providing impetus for further development of targeted nanosystems for localized therapy in future in vivo models. CONCLUSIONS: This work has provided a potential clinical application for targeting GBM CSCs with simultaneous diagnostic imaging.

An Unusual Route for p-Aminobenzoate Biosynthesis in Chlamydia trachomatis Involves a Probable Self-Sacrificing Diiron Oxygenase.

Chlamydia trachomatis lacks the canonical genes required for the biosynthesis of p-aminobenzoate (pABA), a component of essential folate cofactors. Previous studies revealed a single gene from C. trachomatis, the CT610 gene, that rescues Escherichia coli ΔpabA, ΔpabB, and ΔpabC mutants, which are otherwise auxotrophic for pABA. CT610 shares low sequence similarity to nonheme diiron oxygenases, and the previously solved crystal structure revealed a diiron active site. Genetic studies ruled out several potential substrates for CT610-dependent pABA biosynthesis, including chorismate and other shikimate pathway intermediates, leaving the actual precursor(s) unknown. Here, we supplied isotopically labeled potential precursors to E. coli ΔpabA cells expressing CT610 and found that the aromatic portion of tyrosine was highly incorporated into pABA, indicating that tyrosine is a precursor for CT610-dependent pABA biosynthesis. Additionally, in vitro enzymatic experiments revealed that purified CT610 exhibits low pABA synthesis activity under aerobic conditions in the absence of tyrosine or other potential substrates, where only the addition of a reducing agent such as dithiothreitol appears to stimulate pABA production. Furthermore, site-directed mutagenesis studies revealed that two conserved active site tyrosine residues are essential for the pABA synthesis reaction in vitro Thus, the current data are most consistent with CT610 being a unique self-sacrificing enzyme that utilizes its own active site tyrosine residue(s) for pABA biosynthesis in a reaction that requires O2 and a reduced diiron cofactor.IMPORTANCEChlamydia trachomatis is the most reported sexually transmitted infection in the United States and the leading cause of infectious blindness worldwide. Unlike many other intracellular pathogens that have undergone reductive evolution, C. trachomatis is capable of de novo biosynthesis of the essential cofactor tetrahydrofolate using a noncanonical pathway. Here, we identify the biosynthetic precursor to the p-aminobenzoate (pABA) portion of folate in a process that requires the CT610 enzyme from C. trachomatis We further provide evidence that CT610 is a self-sacrificing or "suicide" enzyme that uses its own amino acid residue(s) as the substrate for pABA synthesis. This work provides the foundation for future investigation of this chlamydial pABA synthase, which could lead to new therapeutic strategies for C. trachomatis infections.

Phase 1 study of the MDM2 antagonist RO6839921 in patients with acute myeloid leukemia.

In acute myeloid leukemia (AML), TP53 mutations and dysregulation of wild-type p53 is common and supports an MDM2 antagonist as a therapy. RO6839921 is an inactive pegylated prodrug of the oral MDM2 antagonist idasanutlin (active principle [AP]) that allows for IV administration. This phase 1 monotherapy study evaluated the safety, pharmacokinetics, and pharmacodynamics of RO6839921 in patients with AML. Primary objectives identified dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD). Secondary objectives assessed pharmacokinetic, pharmacodynamic, and antileukemic activity. A total of 26 patients received 120-300 mg AP of idasanutlin. The MTD was 200 mg, with DLTs at 250 (2/8 patients) and 300 mg (2/5). Treatment-related adverse events in >20% of patients were diarrhea, nausea, vomiting, decreased appetite, and fatigue. Six deaths (23.1%) occurred, all unrelated to treatment. Pharmacokinetics showed rapid and near-complete conversion of the prodrug to AP and dose-proportional exposure across doses. Variability ranged from 30%-47% (22%-54% for idasanutlin). TP53 was 21 (87.5%) wild-type and 3 mutant (12.5%). The composite response rate (complete remission [CR], CR with incomplete hematologic recovery/morphological leukemia-free state [CRi/MLFS], or CR without platelet recovery [CRp]) was 7.7%. Antileukemic activity (CR, CRi/MLFS, partial response, hematologic improvement/stable disease) was observed in 11 patients (disease control rate, 42%): 10/11 were TP53 wild-type; 1 had no sample. p53 activation was demonstrated by MIC-1 induction and was associated with AP exposure. There was not sufficient differentiation or improvement in the biologic or safety profile compared with oral idasanutlin to support continued development of RO6839921. NCT02098967.

A phase 1 study of the MDM2 antagonist RO6839921, a pegylated prodrug of idasanutlin, in patients with advanced solid tumors.

Purpose MDM2 is a negative regulator of the tumor suppressor p53. RO6839921 is an inactive pegylated prodrug of idasanutlin, an MDM2 antagonist, developed for intravenous administration. On cleavage by plasma esterases, the active principle (AP = idasanutlin) is released. This phase 1 study investigated the safety, pharmacokinetics, and pharmacodynamics of RO6839921 in patients with advanced solid tumors (NCT02098967). Methods Patients were evaluated on a 5-day dosing schedule every 28 days. Dose escalation used the Bayesian new continual reassessment model. Accelerated dose titration was permitted until grade ≥2 drug-related AEs were observed. The target DLT rate to define the MTD was 16-25%. p53 activation was assessed by measuring macrophage inhibitory cytokine-1 (MIC-1). Results Forty-one patients received 14-120 mg AP; 39 were DLT evaluable. The MTD was 110-mg AP (8% DLT rate), whereas 120-mg AP had a 44% DLT rate. DLTs were neutropenia, thrombocytopenia, and stridor. The most common treatment-related AEs (≥30%) were nausea, fatigue, vomiting, and thrombocytopenia. Pharmacokinetic analyses indicated rapid conversion of prodrug to AP and an approximately linear and dose-proportional dose-exposure relationship, with a 2-fold increase in exposure between Days 1 and 5 of AP. MIC-1 increases were exposure dependent. Stable disease was observed in 14 patients (34%). Conclusions RO6839921 showed reduced pharmacokinetic exposure variability and a safety profile comparable with that of oral idasanutlin. Although this study indicated that RO6839921 could be administered to patients, the results did not provide sufficient differentiation or improvement in the biologic or safety profile compared with oral idasanutlin to support continued development.

Determination of benzonatate and its metabolite in human plasma by HPLC-MS/MS: A preliminary pharmacokinetic study in healthy Chinese volunteers after oral administration of benzonatate soft capsule.

Benzonatate has been used as a non-narcotic oral antitussive drug for many years. Its pharmacokinetics has never been reported due to the technical difficulties in detecting benzonatate by mass spectrometry. However, its concentration can be extrapolated based on the concentration of its metabolite, 4-(butylamino)benzoic acid (BBA). In this study, two sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methods were developed and fully validated for the determination of the original 4-(butylamino)benzoic acid (method B) and total 4-(butylamino)benzoic acid (containing the original 4-(butylamino)benzoic acid and 4-(butylamino)benzoic acid converted from benzonatate after collection, method A). For both methods, one-step protein precipitation by methanol was performed to extract analytes from the plasma samples. Chromatographic separation was done on an InfinityLab Poroshell 120 Phenyl Hexyl column (2.1 mm × 50 mm, 2.7 µm, Agilent) with initial mobile phase consisting of 5 mM ammonium acetate containing 0.3% formic acid and acetonitrile (60:40, v/v) at a flow rate of 0.3 mL/min. Quantification was achieved by multiple reaction monitoring (MRM) in electron spray ionization (ESI) positive mode with the transitions of m/z 194.2 → 138.1 and 515.3 → 497.3 for 4-(butylamino)benzoic acid and telmisartan (the internal standard), respectively. The two methods exhibited good linearity over the concentration range of 10-10000 ng/mL. Both of the methods were successfully applied to the preliminary pharmacokinetic study in healthy Chinese volunteers after oral administration of benzonatate soft capsule at a single dose of 100 mg. The results showed that 4-(butylamino)benzoic acid and benzonatate were rapidly absorbed and reached a maximum concentration (Cmax) of 1708 ±â€¯457 ng/mL and 1063 ±â€¯460 ng/mL, respectively. The half-life (t1/2) were 1.32 ±â€¯0.29 h for 4-(butylamino)benzoic acid and 1.01 ±â€¯0.41 h for benzonatate. The area under the curve from 0 h to 10 h (AUC0-10) for 4-(butylamino)benzoic acid and benzonatate were 2103 ±â€¯918 ng/mL·h and 1097 ±â€¯559 ng/mL·h, respectively. And the data was valuable for further clinical study.

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.

Role of efflux transporters in the absorption, distribution and elimination in rodents of a novel PDE4 inhibitor, CHF6001.

CHF6001 is a new and potent PDE4 inhibitor for the treatment of human lung diseases, designed for topical administration by inhalation. In preclinical assessment CHF6001 appeared safe and devoid of emetic effect, which is typical side effect of PDE4 inhibitors in humans. CHF6001 absorption, distribution and excretion were evaluated in rats by PO and IV administration of [14C]CHF6001; additionally the role of transporters was investigated by using transfected cells expressing either human transporters or MDR1 and BCRP KO mice. [14C]CHF6001 intravenously administered as bolus distributed in all the tissues (with very low levels in brain and fetus) and it was mainly eliminated in bile. Following oral administration [14C]CHF6001 about half of the dose was absorbed through the gut. In vitro, CHF6001 was a substrate of human membrane transporters MDR1 and BCRP. In wild and BCRP KO mice CHF6001 was not detectable in brain, whereas it was measurable in Mdr1a/b KO mice. Therefore, in animal species Mdr1a/b plays a significant role in CHF6001 disposition, limiting its distribution into brain and contributing to the safety profile observed in preclinical evaluation. This behavior was confirmed by the results of the first human studies, where CHF6001 was safe and with no emetic effect at all the evaluated doses.

The effects of dissolved organic matter and feeding on bioconcentration and oxidative stress of ethylhexyl dimethyl p-aminobenzoate (OD-PABA) to crucian carp (Carassius auratus).

Bioconcentration of UV filters in organisms is an important indicator for the assessment of environmental hazards. However, bioconcentration testing rarely accounts for the influence of natural aquatic environmental factors. In order to better assess the ecological risk of organic UV filters (OUV-Fs) in an actual water environment, this study determined the influences of dissolved organic matter (DOM) (0, 1, 10, and 20 mg/L) and feeding (0, 0.5, 1, and 2% body weight/d) on bioconcentration of ethylhexyl dimethyl p-aminobenzoate (OD-PABA) in various tissues of crucian carp (Carassius auratus). Moreover, oxidative stress in the fish liver caused by the OD-PABA was also investigated by measuring activities of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST), and levels of glutathione (GSH) and malondialdehyde (MDA). The bioconcentration of OD-PABA in the fish tissues was significantly decreased with the presence of DOM indicating a reduction of OD-PABA bioavailability caused by DOM. The bioconcentration factors (BCFs) decreased by 28.00~50.93% in the muscle, 72.67~96.74% in the gill, 37.84~87.72% in the liver, and 10.32~79.38% in the kidney at different DOM concentrations compared to those of the non-DOM treatments. Significant changes in SOD, CAT, GST, GSH, and MDA levels were found in the DOM- and OD-PABA-alone treatments. However, there were no significant differences in the SOD, CAT, GST, and MDA levels found when co-exposure to OD-PABA and DOM. Feeding led to lower OD-PABA concentrations in the fish tissues, and the concentrations were decreased with increasing feeding ratios. BCFs in various tissues reduced by 39.75~72.52% in the muscle, 56.86~79.73% in the gill, 66.41~87.50% in the liver, and 75.88~89.10% in the kidney, respectively. In the unfed treatments, the levels of SOD and MDA were significantly higher than those of the fed ones while GST and GSH levels were remarkably inhibited indicating the enhanced effect of starvation to oxidative stress. There was no markedly alternation of the biomarker levels observed between different fed treatments. In conclusion, our study indicated that both DOM and feeding reduced bioconcentration of OD-PABA and alleviated oxidative stress to some extent in the crucian carp.

Metabolic crosstalk regulates Porphyromonas gingivalis colonization and virulence during oral polymicrobial infection.

Many human infections are polymicrobial in origin, and interactions among community inhabitants shape colonization patterns and pathogenic potential 1 . Periodontitis, which is the sixth most prevalent infectious disease worldwide 2 , ensues from the action of dysbiotic polymicrobial communities 3 . The keystone pathogen Porphyromonas gingivalis and the accessory pathogen Streptococcus gordonii interact to form communities in vitro and exhibit increased fitness in vivo 3,4 . The mechanistic basis of this polymicrobial synergy, however, has not been fully elucidated. Here we show that streptococcal 4-aminobenzoate/para-amino benzoic acid (pABA) is required for maximal accumulation of P. gingivalis in dual-species communities. Metabolomic and proteomic data showed that exogenous pABA is used for folate biosynthesis, and leads to decreased stress and elevated expression of fimbrial adhesins. Moreover, pABA increased the colonization and survival of P. gingivalis in a murine oral infection model. However, pABA also caused a reduction in virulence in vivo and suppressed extracellular polysaccharide production by P. gingivalis. Collectively, these data reveal a multidimensional aspect to P. gingivalis-S. gordonii interactions and establish pABA as a critical cue produced by a partner species that enhances the fitness of P. gingivalis while diminishing its virulence.

Fabrication of autofluorescent porous silica nanoparticles for redox-responsive drug release.

Porous silica nanoparticles were prepared by emulsion-condensation route. The silica nanoparticles with diameter of 50nm have both accessible center-radial large pore channels (19.9nm) and small pore size of 3.5nm. The hierarchical porous structure endows them large pore volume for loading drugs and sustained release property. The silica nanoparticles were further modified with glucose-oxidized glutathione. The formulated Schiff base and disulfide bonds render the silica nanoparticles auto-fluorescent and redox-responsive properties. The cleavage of disulfide bonds caused by reactive thiols facilitates aminomethylbenzoic acid (AMA) release. The release of drug leads to the loss of fluorescence, which would be used to monitor the drug delivery and carrier distribution.

Production of para-aminobenzoate by genetically engineered Corynebacterium glutamicum and non-biological formation of an N-glucosyl byproduct.

para-Aminobenzoate (PABA), a valuable chemical raw material, can be synthesized by most microorganisms. This aromatic compound is currently manufactured from petroleum-derived materials by chemical synthesis. To produce PABA from renewable resources, its production by fermentation was investigated. The evaluation of the sensitivity to PABA toxicity revealed that Corynebacterium glutamicum had better tolerance to PABA than several other microorganisms. To produce PABA from glucose, genetically engineered C. glutamicum was constructed by introducing both pabAB and pabC. The generated strain produced 20mM of PABA in a test-tube scale culture; however, during the investigation, an unidentified major byproduct was detected in the culture supernatant. Unexpectedly, the byproduct was also detected after the incubation of PABA with glucose in a buffer solution without bacterial cells. To elucidate the mechanism underlying the formation of this byproduct, PABA analogues and several kinds of sugars were mixed and analyzed. New chemical compounds were detected when incubating aniline with glucose as well as PABA with reducing sugars (mannose, xylose, or arabinose), indicating that an amino group of PABA reacted non-enzymatically with an aldehyde group of glucose. The molecular mass of the byproduct determined by LC-MS suggested that the molecule was generated from PABA and glucose with releasing a water molecule, generally known as a glycation product. Because the glycation reaction was reversible, the byproduct was easily converted to PABA by acid treatment (around pH 2-3) with HCl. Then, pab genes were screened to improve PABA production. The highest PABA concentration was achieved by a strain expressing the pabAB of Corynebacterium callunae and a strain expressing the pabC of Xenorhabdus bovienii, respectively. A plasmid harboring both the pabAB of C. callunae and the pabC of X. bovienii, the best gene combination, was introduced into a strain overexpressing the genes of the shikimate pathway. The resultant strain produced 45mM of PABA in a test-tube scale culture. Under a fermenter-controlled condition, the strain produced up to 314mM (43g/L) of PABA at 48h, with a 20% yield. To our knowledge, this is the highest concentration of PABA produced by a genetically modified microorganism ever reported.

Investigating the effect of autoinduction in cynomolgus monkeys of a novel anticancer MDM2 antagonist, idasanutlin, and relevance to humans.

1. Idasanutlin (RG7388) is a potent p53-MDM2 antagonist currently in clinical development for treatment of cancer. The purpose of the present studies was to investigate the cause of marked decrease in plasma exposure after repeated oral administration of RG7388 in monkeys and whether the autoinduction observed in monkeys is relevant to humans. 2. In monkey liver and intestinal microsomes collected after repeated oral administration of RG7388 to monkeys, significantly increased activities of homologue CYP3A8 were observed (ex vivo). Investigation using a physiologically based pharmacokinetic (PBPK) model suggested that the loss of exposure was primarily due to induction of metabolism in the gut of monkeys. 3. Studies in monkey and human primary hepatocytes showed that CYP3A induction by RG7388 only occurred in monkey hepatocytes but not in human hepatocytes, which suggests the observed CYP3A induction is monkey specific. 4. The human PK data obtained from the first cohorts confirmed the lack of relevant induction as predicted by the human hepatocytes and the PBPK modelling based on no induction in humans.

Non-stabilized nucleophiles in Cu-catalysed dynamic kinetic asymmetric allylic alkylation.

The development of new reactions forming asymmetric carbon-carbon bonds has enabled chemists to synthesize a broad range of important carbon-containing molecules, including pharmaceutical agents, fragrances and polymers. Most strategies to obtain enantiomerically enriched molecules rely on either generating new stereogenic centres from prochiral substrates or resolving racemic mixtures of enantiomers. An alternative strategy--dynamic kinetic asymmetric transformation--involves the transformation of a racemic starting material into a single enantiomer product, with greater than 50 per cent maximum yield. The use of stabilized nucleophiles (pKa < 25, where Ka is the acid dissociation constant) in palladium-catalysed asymmetric allylic alkylation reactions has proved to be extremely versatile in these processes. Conversely, the use of non-stabilized nucleophiles in such reactions is difficult and remains a key challenge. Here we report a copper-catalysed dynamic kinetic asymmetric transformation using racemic substrates and alkyl nucleophiles. These nucleophiles have a pKa of ≥50, more than 25 orders of magnitude more basic than the nucleophiles that are typically used in such transformations. Organometallic reagents are generated in situ from alkenes by hydrometallation and give highly enantioenriched products under mild reaction conditions. The method is used to synthesize natural products that possess activity against tuberculosis and leprosy, and an inhibitor of para-aminobenzoate biosynthesis. Mechanistic studies indicate that the reaction proceeds through a rapidly isomerizing intermediate. We anticipate that this approach will be a valuable complement to existing asymmetric catalytic methods.

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.

Enhancing pterin and para-aminobenzoate content is not sufficient to successfully biofortify potato tubers and Arabidopsis thaliana plants with folate.

Folates are important cofactors in one-carbon metabolism in all living organisms. Since only plants and micro- organisms are capable of biosynthesizing folates, humans depend entirely on their diet as a folate source. Given the low folate content of several staple crop products, folate deficiency affects regions all over the world. Folate biofortification of staple crops through enhancement of pterin and para-aminobenzoate levels, precursors of the folate biosynthesis pathway, was reported to be successful in tomato and rice. This study shows that the same strategy is not sufficient to enhance folate content in potato tubers and Arabidopsis thaliana plants and concludes that other steps in folate biosynthesis and/or metabolism need to be engineered to result in substantial folate accumulation. The findings provide a plausible explanation why, more than half a decade after the proof of concept in rice and tomato, successful folate biofortification of other food crops through enhancement of para-aminobenzoate and pterin content has not been reported thus far. A better understanding of the folate pathway is required in order to determine an engineering strategy that can be generalized to most staple crops.

Completing the folate biosynthesis pathway in Plasmodium falciparum: p-aminobenzoate is produced by a highly divergent promiscuous aminodeoxychorismate lyase.

Enzymes that produce or recycle folates are the targets of widely used antimalarial drugs. Despite the interest in the folate metabolism of Plasmodium falciparum, the molecular identification of ADCL (aminodeoxychorismate lyase), which synthesizes the p-aminobenzoate moiety of folate, remained unresolved. In the present study, we demonstrate that the plasmodial gene PF14_0557 encodes a functional ADCL and report a characterization of the recombinant enzyme.

High-throughput screening AlphaScreen assay for identification of small-molecule inhibitors of ubiquitin E3 ligase SCFSkp2-Cks1.

Decreased levels of cell cycle inhibitor p27(Kip1) due to excessive degradation occur in a variety of aggressive human tumors. Since reduced p27(Kip1) expression has been associated with a poor prognosis in many human cancers and resistance to certain antitumor therapies, elevation of p27(Kip1) expression could improve prognosis and prevent excessive cell proliferation. SCF(Skp2) is one of the major ubiquitin E3 ligases responsible for degradation of p27(Kip1). Ubiquitination of p27(Kip1) also requires a small adaptor protein, Cks1, which facilitates substrate recruitment by bridging the interaction between Skp2 and p27(Kip1). It has been shown previously that a direct interaction between Cks1 and Skp2 is required for p27(Kip1) degradation. Accordingly, perturbation of the Skp2-Cks1 interaction may represent an attractive target for pharmacological intervention. Here we describe a high-throughput AlphaScreen assay for discovering small-molecule inhibitors of the Skp2-Cks1 protein-protein interaction in vitro. Two compounds (NSC689857 and NSC681152) were identified and validated through a structure-activity relationship analysis. Both compounds were also shown to inhibit p27(Kip1) ubiquitination in vitro. These studies demonstrate that disruption of the Skp2-Cks1 interaction provides a viable strategy to prevent p27(Kip1) ubiquitination and may potentially be useful for the control of excessive degradation of this cell cycle inhibitor in tumor cells.