Probing the origins of glutathione biosynthesis through biochemical analysis of glutamate-cysteine ligase and glutathione synthetase from a model photosynthetic prokaryote.

William B Musgrave, Hankuil Yi, Dustin Kline, Jeffrey C Cameron, Jonathan Wignes, Sanghamitra Dey, Himadri B Pakrasi, Joseph M Jez

Department of Biology, Washington University, One Brookings Drive, Campus Box 1137, St. Louis, MO 63130, USA.

The Biochemical journal 2013 Feb 15

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Glutathione biosynthesis catalysed by GCL (glutamate-cysteine ligase) and GS (glutathione synthetase) is essential for maintaining redox homoeostasis and protection against oxidative damage in diverse eukaroytes and bacteria. This biosynthetic pathway probably evolved in cyanobacteria with the advent of oxygenic photosynthesis, but the biochemical characteristics of progenitor GCLs and GSs in these organisms are largely unexplored. In the present study we examined SynGCL and SynGS from Synechocystis sp. PCC 6803 using steady-state kinetics. Although SynGCL shares ~15% sequence identity with the enzyme from plants and α-proteobacteria, sequence comparison suggests that these enzymes share similar active site residues. Biochemically, SynGCL lacks the redox regulation associated with the plant enzymes and functions as a monomeric protein, indicating that evolution of redox regulation occurred later in the green lineage. Site-directed mutagenesis of SynGCL establishes this enzyme as part of the plant-like GCL family and identifies a catalytically essential arginine residue, which is structurally conserved across all forms of GCLs, including those from non-plant eukaryotes and γ-proteobacteria. A reaction mechanism for the synthesis of γ-glutamylcysteine by GCLs is proposed. Biochemical and kinetic analysis of SynGS reveals that this enzyme shares properties with other prokaryotic GSs. Initial velocity and product inhibition studies used to examine the kinetic mechanism of SynGS suggest that it and other prokaryotic GSs uses a random ter-reactant mechanism for the synthesis of glutathione. The present study provides new insight on the molecular mechanisms and evolution of glutathione biosynthesis; a key process required for enhancing bioenergy production in photosynthetic organisms.

Citation

William B Musgrave, Hankuil Yi, Dustin Kline, Jeffrey C Cameron, Jonathan Wignes, Sanghamitra Dey, Himadri B Pakrasi, Joseph M Jez. Probing the origins of glutathione biosynthesis through biochemical analysis of glutamate-cysteine ligase and glutathione synthetase from a model photosynthetic prokaryote. The Biochemical journal. 2013 Feb 15;450(1):63-72