Ophthalmic Acid

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.

Background and aim: Animal studies indicated that systemic ophthalmic acid (OPH) is a biomarker for hepatic glutathione (GSH) homeostasis, an important determinant of liver function. We aimed to clarify whether OPH levels can be used as a read-out for hepatic GSH homeostasis after paracetamol (APAP) challenges during pylorus-preserving pancreaticoduodenectomy (PPPD) or partial hepatectomy (PH). Methods: Nineteen patients undergoing PPPD (n=7, control group) or PH (n=12) were included. APAP (1000 mg) was administered intravenously before resection (first challenge), and six and twelve hours later, with sequential blood sampling during this period. Arterial, hepatic and portal venous blood samples and liver biopsies were taken on three occasions during the first APAP challenge. Plasma and hepatic OPH and GSH levels were quantified, and venous-arterial differences were calculated to study hepatic release. Results: Systemic GSH levels decreased during the course of the APAP challenge in both surgical groups, without notable change in OPH levels. Hepatic GSH and OPH content was not affected within ˜3 hours after administration of the first APAP dose in patients undergoing PPPD or PH. In this period, net release of OPH by the liver was observed only in patients undergoing PPPD. Conclusion: The drop in circulating GSH levels following APAP administrations, did not result in an increase in plasma OPH in both patients with an intact liver and in those undergoing liver resection. Hepatic content of GSH and OPH was not affected during the first APAP dose. It is uncertain whether hepatic GSH homeostasis was sufficiently challenged in the present study. Relevance for patients: In the present study, plasma OPH seemed not useful as a marker for GSH depletion because APAP administration during liver surgery did not lead to (immediate) GSH depletion or increased OPH levels. Based on stable levels of hepatic GSH, OPH and thiyl radicals during surgery, standard APAP administration seems to be safe in a postoperative care program with regards to GSH homeostasis in this specific population. However, no general statements can be made on the basis of the current experiment, since GSH homeostasis and susceptibility to xenobiotic toxicity are influenced by several metabolic and genetic factors.

Background: Ophthalmic acid (OPH), γ-glutamyl-L-2-aminobutyryl-glycine, a tripeptide analogue of glutathione (GSH), has recently captured considerable attention as a biomarker of oxidative stress in animals. The OPH and GSH biosynthesis, as well as some biochemical behaviors, are very similar. Here, we sought to investigate the presence of OPH in plants and its possible relationship with GSH, known to possess multiple functions in the plant development, growth and response to environmental changes. Methods: HPLC-ESI-MS/MS analysis was used to examine the occurrence of OPH in leaves from various plant species, and flours from several plant seeds. Different types of oxidative stress, i.e., water, dark, paraquat, and cadmium stress, were induced in rye, barley, oat, and winter wheat leaves to evaluate the effects on the levels of OPH and its metabolic precursors. Results: OPH and its dipeptide precursor, γ-glutamyl-2-aminobutyric acid, were found to occur in phylogenetically distant plants. Interestingly, the levels of OPH were tightly associated with the oxidative stress tested. Levels of OPH precursors, γ-glutamyl-2-aminobutyric acid and 2-aminobutyric acid, the latter efficiently formed in plants via biosynthetic pathways absent in the animal kingdom, were also found to increase during oxidative stress. Conclusions: OPH occurs in plants and its levels are tightly associated with oxidative stress. General significance: OPH behaves as an oxidative stress marker and its biogenesis might occur through a biochemical pathway common to many living organisms.