Characterization of the bifunctional <tex>$\gamma$</tex>-glutamate-cysteine ligase/glutathione synthetase (GshF) of **Pasteurella multocida**
Faculty of Sciences. Biology
Journal of biological chemistry. - Baltimore, Md
, p. 4380-4394
Glutamate-cysteine ligase (γ-ECL) and glutathione synthetase (GS) are the two unrelated ligases that constitute the glutathione biosynthesis pathway in most eukaryotes, purple bacteria, and cyanobacteria. γ-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 γ-proteobacterial γ-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 ∼85-kDa protein, which, on the basis of gel-sizing chromatography, forms dimers in solution. The γ-ECL activity of GshF is regulated by an allosteric type of glutathione feedback inhibition (Ki = 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.