title: Ophthalmic acid accumulation in an Escherichia coli mutant lacking the conserved pyridoxal 5'-phosphate-binding protein YggS.
abstract: Escherichia coli YggS is a highly conserved pyridoxal 5'-phosphate (PLP)-binding protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/MS and MS/MS analyses of the compound with propyl chloroformate derivatization, and co-chromatography analysis identified this compound as γ-l-glutamyl-l-2-aminobutyryl-glycine (ophthalmic acid), a glutathione (GSH) analogue in which the l-Cys moiety is replaced by l-2-AB. We also determine the metabolic consequence of the yggS mutation. Absence of YggS initially increases l-2-AB availability, and then causes ophthalmic acid accumulation and CoA limitation in the cell. The expression of a γ-glutamylcysteine synthetase and a glutathione synthetase in a ΔyggS background causes high-level accumulation of ophthalmic acid in the cells (∼1.2 nmol/mg cells) in a minimal synthetic medium. This opens the possibility of a first fermentative production of ophthalmic acid.
authors.affiliation: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furou-chou, Chikusa, Nagoya, Aichi 464-8601, Japan. Electronic address: ito-t@agr.nagoya-u.ac.jp.
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Ito
authors.affiliation: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furou-chou, Chikusa, Nagoya, Aichi 464-8601, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Yamauchi
authors.affiliation: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furou-chou, Chikusa, Nagoya, Aichi 464-8601, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hemmi
authors.affiliation: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furou-chou, Chikusa, Nagoya, Aichi 464-8601, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Yoshimura
mesh_terms: D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003065:Coenzyme A / Q000378:metabolism; D017124:Conserved Sequence; D004151:Dipeptides / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism; D005981:Glutathione Synthase / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000096:biosynthesis*; D030781:Organisms, Genetically Modified; D028044:Phosphate-Binding Proteins / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D002352:Carrier Proteins; D004151:Dipeptides; D029968:Escherichia coli Proteins; D009842:Oligopeptides; D028044:Phosphate-Binding Proteins; C585834:YggS protein, E coli; C016632:ophthalmic acid; D005721:Glutamate-Cysteine Ligase; D005981:Glutathione Synthase; C017341:gamma-glutamylcysteine; D003065:Coenzyme A
keywords: Amino acid; Escherichia coli; Glutathione; Ophthalmic acid; Pyridoxal 5′-phosphate; YggS