Characterization of the bifunctional gamma-glutamate-cysteine ligase/glutathione synthetase (GshF) of Pasteurella multocida.
Glutamate-cysteine ligase (gamma-ECL) and glutathione synthetase (GS) are the two unrelated ligases that constitute the glutathione biosynthesis pathway in most eukaryotes, purple bacteria, and cyanobacteria. gamma-ECL is a member of the glutamine synthetase family, whereas GS enzymes group together with highly diverse carboxyl-to-amine/thiol ligases, all characterized by the so-called two-domain ATP-grasp fold. This generalized scheme toward the formation of glutathione, however, is incomplete, as functional steady-state levels of intracellular glutathione may also accumulate solely by import, as has been reported for the Pasteurellaceae member Haemophilus influenzae, as well as for certain Gram-positive enterococci and streptococci, or by the action of a bifunctional fusion protein (termed GshF), as has been reported recently for the Gram-positive firmicutes Streptococcus agalactiae and Listeria monocytogenes. Here, we show that yet another member of the Pasteurellaceae family, Pasteurella multocida, acquires glutathione both by import and GshF-driven biosynthesis. Domain architecture analysis shows that this P. multocida GshF bifunctional ligase contains an N-terminal gamma-proteobacterial gamma-ECL-like domain followed by a typical ATP-grasp domain, which most closely resembles that of cyanophycin synthetases, although it has no significant homology with known GS ligases. Recombinant P. multocida GshF overexpresses as an approximately 85-kDa protein, which, on the basis of gel-sizing chromatography, forms dimers in solution. The gamma-ECL activity of GshF is regulated by an allosteric type of glutathione feedback inhibition (K(i) = 13.6 mM). Furthermore, steady-state kinetics, on the basis of which we present a novel variant of half-of-the-sites reactivity, indicate intimate domain-domain interactions, which may explain the bifunctionality of GshF proteins.
Vergauwen
Bjorn
B
Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, Belgium.
De Vos
Dirk
D
Van Beeumen
Jozef J
JJ
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics; D005721:Glutamate-Cysteine Ligase / Q000737:chemistry / Q000502:physiology*; D005978:Glutathione / Q000096:biosynthesis; D005981:Glutathione Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D016979:Pasteurella multocida / Q000201:enzymology*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D005721:Glutamate-Cysteine Ligase; D005981:Glutathione Synthase; D005978:Glutathione