PMID:16339152

Citation	Vergauwen, B, De Vos, D and Van Beeumen, JJ (2006) Characterization of the bifunctional gamma-glutamate-cysteine ligase/glutathione synthetase (GshF) of Pasteurella multocida. J. Biol. Chem. 281:4380-94
Abstract	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.
Links	PubMed Online version:10.1074/jbc.M509517200
Keywords	Amino Acid Sequence; Escherichia coli/genetics; Glutamate-Cysteine Ligase/chemistry; Glutamate-Cysteine Ligase/physiology; Glutathione/biosynthesis; Glutathione Synthase/antagonists & inhibitors; Glutathione Synthase/chemistry; Glutathione Synthase/physiology; Molecular Sequence Data; Oxidative Stress; Pasteurella multocida/enzymology; Protein Structure, Secondary; Protein Structure, Tertiary
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
PASMU:GSHAB	Contributes to	GO:0004357: glutamate-cysteine ligase activity	ECO:0000314:		F		Table 2 shows an enzyme assay used in order to demonstrate Glutamate-cysteine ligase formation from GshF	complete CACAO 4592

References

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