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A Multidomain Fusion Protein in Listeria monocytogenes Catalyzes the Two Primary Activities for Glutathione Biosynthesis

Shubha Gopal,^2, Ilya Borovok,¹ Amos Ofer,¹ Michaela Yanku,¹ Gerald Cohen,¹ Werner Goebel,² Jürgen Kreft,² and Yair Aharonowitz^1*

Tel Aviv University, The George S. Wise Faculty of Life Sciences, Department of Molecular Microbiology and Biotechnology, Ramat Aviv, 69978, Tel Aviv, Israel,¹ Theodor-Boveri-Institut (Biozentrum) der Universitat Würzburg, Lehrstuhl für Mikrobiologie, Am Hubland, 97074 Würzburg, Germany²

Received 16 January 2005/ Accepted 24 February 2005

Glutathione is the predominant low-molecular-weight peptide thiol present in living organisms and plays a key role in protecting cells against oxygen toxicity. Until now, glutathione synthesis was thought to occur solely through the consecutive action of two physically separate enzymes, -glutamylcysteine ligase and glutathione synthetase. In this report we demonstrate that Listeria monocytogenes contains a novel multidomain protein (termed GshF) that carries out complete synthesis of glutathione. Evidence for this comes from experiments which showed that in vitro recombinant GshF directs the formation of glutathione from its constituent amino acids and the in vivo effect of a mutation in GshF that abolishes glutathione synthesis, results in accumulation of the intermediate -glutamylcysteine, and causes hypersensitivity to oxidative agents. We identified GshF orthologs, consisting of a -glutamylcysteine ligase (GshA) domain fused to an ATP-grasp domain, in 20 gram-positive and gram-negative bacteria. Remarkably, 95% of these bacteria are mammalian pathogens. A plausible origin for GshF-dependent glutathione biosynthesis in these bacteria was the recruitment by a GshA ancestor gene of an ATP-grasp gene and the subsequent spread of the fusion gene between mammalian hosts, most likely by horizontal gene transfer.

Present address: Department of Biotechnology, P.A. College of Engineering, Nadupav, Kairangala, Near Mangalore University, Mangalore 574153, India.

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