Plant glutathione biosynthesis revisited: redox-mediated activation of glutamylcysteine ligase does not require homo-dimerization

Biochem J. 2019 Apr 15;476(7):1191-1203. doi: 10.1042/BCJ20190072.

Abstract

Plant γ-glutamylcysteine ligase (GCL), catalyzing the first and tightly regulated step of glutathione (GSH) biosynthesis, is redox-activated via formation of an intramolecular disulfide bond. In vitro, redox-activation of recombinant GCL protein causes formation of homo-dimers. Here, we have investigated whether dimerization occurs in vivo and if so whether it contributes to redox-activation. FPLC analysis indicated that recombinant redox-activated WT (wild type) AtGCL dissociates into monomers at concentrations below 10-6 M, i.e. below the endogenous AtGCL concentration in plastids, which was estimated to be in the micromolar range. Thus, dimerization of redox-activated GCL is expected to occur in vivo To determine the possible impact of dimerization on redox-activation, AtGCL mutants were generated in which salt bridges or hydrophobic interactions at the dimer interface were interrupted. WT AtGCL and mutant proteins were analyzed by non-reducing SDS-PAGE to address their redox state and probed by FPLC for dimerization status. Furthermore, their substrate kinetics (K M, V max) were compared. The results indicate that dimer formation is not required for redox-mediated enzyme activation. Also, crystal structure analysis confirmed that dimer formation does not affect binding of GSH as competitive inhibitor. Whether dimerization affects other enzyme properties, e.g. GCL stability in vivo, remains to be investigated.

Keywords: glutamylcysteine ligase; glutathione biosynthesis; homo-dimerization; redox activation; site-directed mutagenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Arabidopsis / genetics
  • Arabidopsis / metabolism
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism*
  • Catalytic Domain
  • Enzyme Activation
  • Glutamate-Cysteine Ligase / chemistry*
  • Glutamate-Cysteine Ligase / genetics
  • Glutamate-Cysteine Ligase / metabolism*
  • Glutathione / biosynthesis*
  • Kinetics
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Oxidation-Reduction
  • Plants, Genetically Modified
  • Protein Multimerization
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism

Substances

  • Arabidopsis Proteins
  • Recombinant Proteins
  • Glutamate-Cysteine Ligase
  • Glutathione