Inhibition of glycine cleavage system by pyridoxine 5'-phosphate causes synthetic lethality in glyA yggS and serA yggS in Escherichia coli.

The YggS/Ybl036c/PLPBP family includes conserved pyridoxal 5'-phosphate (PLP)-binding proteins that play a critical role in the homeostasis of vitamin B6 and amino acids. Disruption of members of this family causes pleiotropic effects in many organisms by unknown mechanisms. In Escherichia coli, conditional lethality of the yggS and glyA (encoding serine hydroxymethyltransferase) has been described, but the mechanism of lethality was not determined. Strains lacking yggS and serA (3-phosphoglycerate dehydrogenase) were conditionally lethality in the M9-glucose medium supplemented with Gly. Analyses of vitamin B6 pools found the high-levels of pyridoxine 5'-phosphate (PNP) in the two yggS mutants. Growth defects of the double mutants could be eliminated by overexpressing PNP/PMP oxidase (PdxH) to decrease the PNP levels. Further, a serA pdxH strain, which accumulates PNP in the presence of yggS, exhibited similar phenotype to serA yggS mutant. Together these data suggested the inhibition of the glycine cleavage (GCV) system caused the synthetic lethality. Biochemical assays confirmed that PNP disrupts the GCV system by competing with PLP in GcvP protein. Our data are consistent with a model in which PNP-dependent inhibition of the GCV system causes the conditional lethality observed in the glyA yggS or serA yggS mutants.

Conserved Pyridoxal 5'-Phosphate-Binding Protein YggS Impacts Amino Acid Metabolism through Pyridoxine 5'-Phosphate in Escherichia coli.

Escherichia coli YggS (COG0325) is a member of the highly conserved pyridoxal 5'-phosphate (PLP)-binding protein (PLPBP) family. Recent studies suggested a role for this protein family in the homeostasis of vitamin B6 and amino acids. The deletion or mutation of a member of this protein family causes pleiotropic effects in many organisms and is causative of vitamin B6-dependent epilepsy in humans. To date, little has been known about the mechanism by which lack of YggS results in these diverse phenotypes. In this study, we determined that the pyridoxine (PN) sensitivity observed in yggS-deficient E. coli was caused by the pyridoxine 5'-phosphate (PNP)-dependent overproduction of Val, which is toxic to E. coli The data suggest that the yggS mutation impacts Val accumulation by perturbing the biosynthetic of Thr from homoserine (Hse). Exogenous Hse inhibited the growth of the yggS mutant, caused further accumulation of PNP, and increased the levels of some intermediates in the Thr-Ile-Val metabolic pathways. Blocking the Thr biosynthetic pathway or decreasing the intracellular PNP levels abolished the perturbations of amino acid metabolism caused by the exogenous PN and Hse. Our data showed that a high concentration of intracellular PNP is the root cause of at least some of the pleiotropic phenotypes described for a yggS mutant of E. coliIMPORTANCE Recent studies showed that deletion or mutation of members of the YggS protein family causes pleiotropic effects in many organisms. Little is known about the causes, mechanisms, and consequences of these diverse phenotypes. It was previously shown that yggS mutations in E. coli result in the accumulation of PNP and some metabolites in the Ile/Val biosynthetic pathway. This work revealed that some exogenous stresses increase the aberrant accumulation of PNP in the yggS mutant. In addition, the current report provides evidence indicating that some, but not all, of the phenotypes of the yggS mutant in E. coli are due to the elevated PNP level. These results will contribute to continuing efforts to determine the molecular functions of the members of the YggS protein family.

The discovery and characterization of K-563, a novel inhibitor of the Keap1/Nrf2 pathway produced by Streptomyces sp.

Keap1/Nrf2 pathway regulates the antioxidant stress response, detoxification response, and energy metabolism. Previous reports found that aberrant Keap1/Nrf2 pathway activation due to Kelch-like ECH-associated protein 1 (Keap1) mutations or Nuclear factor E2-related factor 2 (Nrf2) mutations induced resistance of cancer cells to chemotherapy and accelerated cell growth via the supply of nutrients. Therefore, Keap1/Nrf2 pathway activation is associated with a poor prognosis in many cancers. These previous findings suggested that inhibition of Keap1/Nrf2 pathway could be a target for anti-cancer therapies. To discover a small-molecule Keap1/Nrf2 pathway inhibitor, we conducted high-throughput screening in Keap1 mutant human lung cancer A549 cells using a transcriptional reporter assay. Through this screening, we identified the novel Keap1/Nrf2 pathway inhibitor K-563, which was isolated from actinomycete Streptomyces sp. K-563 suppressed the expression of Keap1/Nrf2 pathway downstream target genes or the downstream target protein, which induced suppression of GSH production, and activated reactive oxygen species production in A549 cells. K-563 also inhibited the expression of downstream target genes in other Keap1- or Nrf2-mutated cancer cells. Furthermore, K-563 exerted anti-proliferative activities in these mutated cancer cells. These in vitro analyses showed that K-563 was able to inhibit cell growth in Keap1- or Nrf2-mutated cancer cells by Keap1/Nrf2 pathway inhibition. K-563 also exerted synergistic combinational effects with lung cancer chemotherapeutic agents. An in vivo study in mice xenotransplanted with A549 cells to further explore the therapeutic potential of K-563 revealed that it also inhibited Keap1/Nrf2 pathway in lung cancer tumors. K-563, a novel Keap1/Nrf2 pathway inhibitor, may be a lead compound for development as an anti-cancer agent.

Production of Ophthalmic Acid Using Engineered Escherichia coli.

Ophthalmic acid (OA; l-γ-glutamyl-l-2-aminobutyryl-glycine) is an analog of glutathione (GSH; l-γ-glutamyl-l-cysteinyl-glycine) in which the cysteine moiety is replaced by l-2-aminobutyrate. OA is a useful peptide for the pharmaceutical and/or food industries. Herein, we report a method for the production of OA using engineered Escherichia coli cells. yggS-deficient E. coli, which lacks the highly conserved pyridoxal 5'-phosphate-binding protein YggS and naturally accumulates OA, was selected as the starting strain. To increase the production of OA, we overexpressed the OA biosynthetic enzymes glutamate-cysteine ligase (GshA) and glutathione synthase (GshB), desensitized the product inhibition of GshA, and eliminated the OA catabolic enzyme γ-glutamyltranspeptidase. The production of OA was further enhanced by the deletion of miaA and ridA with the aim of increasing the availability of ATP and attenuating the unwanted degradation of amino acids, respectively. The final strain developed in this study successfully produced 277 µmol/liter of OA in 24 h without the formation of by-products in a minimal synthetic medium containing 1 mM each glutamate, 2-aminobutyrate, and glycine.IMPORTANCE Ophthalmic acid (OA) is a peptide that has the potential for use in the pharmaceutical and/or food industries. An efficient method for the production of OA would allow us to expand our knowledge about its physiological functions and enable the industrial/pharmaceutical application of this compound. We demonstrated the production of OA using Escherichia coli cells in which OA biosynthetic enzymes and degradation enymes were engineered. We also showed that unique approaches, including the use of a ΔyggS mutant as a starting strain, the establishment of an S495F mutation in GshA, and the deletion of ridA or miaA, facilitated the efficient production of OA in E. coli.

The synthesis and evaluation of the antiproliferative activity of deacidified GEX1A analogues.

GEX1A/herboxidiene (1) is a natural product isolated from Streptomyces sp. and has been reported to target the pre-mRNA splicing process. Although 1 was shown to have antitumor activity in vivo, weight loss was observed in mice when 1 was consecutively administered. We assumed that the carboxylic acid moiety was one of the causes of this toxicity. In this study, a series of amide, carbamate and urea analogues of 1 were synthesized and their antiproliferative activity was evaluated in vitro. The synthesis of urea analogues featured Curtius rearrangement following amine treatment with the one-pot procedure from 1. Furthermore, a structure-activity relationship study of the urea analogues revealed that the pharmacologically preferable basic side chains were acceptable and that compound 9g was equipotent to parent 1. These basic urea analogues would be promising leads for the development of novel antitumor agents.