Characterization of the bifunctional <script type="math/tex">\gamma</script>-glutamate-cysteine ligase/glutathione synthetase (GshF) of **Pasteurella multocida**

Vergauwen, Bjorn; De Vos, Dirk; Van Beeumen, Jozef J.

doi:10.1074/JBC.M509517200

Title

Characterization of the bifunctional $\gamma$ -glutamate-cysteine ligase/glutathione synthetase (GshF) of **Pasteurella multocida**

Author

Vergauwen, Bjorn

De Vos, Dirk

Van Beeumen, Jozef J.

Abstract

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.

Language

English

Source (journal)

Journal of biological chemistry. - Baltimore, Md

Publication

Baltimore, Md : 2006

ISSN

0021-9258 [print]

1083-351X [online]

DOI

10.1074/JBC.M509517200

Volume/pages