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J. Biol. Chem., Vol. 281, Issue 7, 4380-4394, February 17, 2006

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Characterization of the Bifunctional -Glutamate-cysteine Ligase/Glutathione Synthetase (GshF) of Pasteurella multocida^*

From the Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, K. L. Ledeganckstraat 35, 9000 Gent, Belgium

Glutamate-cysteine ligase (-ECL) and glutathione synthetase (GS) are the two unrelated ligases that constitute the glutathione biosynthesis pathway in most eukaryotes, purple bacteria, and cyanobacteria. -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 -proteobacterial -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 -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.

Received for publication, August 29, 2005 , and in revised form, December 6, 2005.

^* This work was supported by Grant 3G003601 from the Fonds of Wetenschappelijk Onderzoek Vlaanderen. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 32-9-264-51-27; Fax: 32-9-264-53-38; E-mail: Jozef.Vanbeeumen{at}Ugent.be.

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