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J. Biol. Chem., Vol. 283, Issue 25, 17672-17680, June 20, 2008

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Leishmania Trypanothione Synthetase-Amidase Structure Reveals a Basis for Regulation of Conflicting Synthetic and Hydrolytic Activities^*

From the Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom

The bifunctional trypanothione synthetase-amidase catalyzes biosynthesis and hydrolysis of the glutathione-spermidine adduct trypanothione, the principal intracellular thiol-redox metabolite in parasitic trypanosomatids. These parasites are unique with regard to their reliance on trypanothione to determine intracellular thiol-redox balance in defense against oxidative and chemical stress and to regulate polyamine levels. Enzymes involved in trypanothione biosynthesis provide essential biological activities, and those absent from humans or for which orthologues are sufficiently distinct are attractive targets to underpin anti-parasitic drug discovery. The structure of Leishmania major trypanothione synthetase-amidase, determined in three crystal forms, reveals two catalytic domains. The N-terminal domain, a cysteine, histidine-dependent amidohydrolase/peptidase amidase, is a papain-like cysteine protease, and the C-terminal synthetase domain displays an ATP-grasp family fold common to C:N ligases. Modeling of substrates into each active site provides insight into the specificity and reactivity of this unusual enzyme, which is able to catalyze four reactions. The domain orientation is distinct from that observed in a related bacterial glutathionylspermidine synthetase. In trypanothione synthetase-amidase, the interactions formed by the C terminus, binding in and restricting access to the amidase active site, suggest that the balance of ligation and hydrolytic activity is directly influenced by the alignment of the domains with respect to each other and implicate conformational changes with amidase activity. The potential inhibitory role of the C terminus provides a mechanism to control relative levels of the critical metabolites, trypanothione, glutathionylspermidine, and spermidine in Leishmania.

Received for publication, March 6, 2008 , and in revised form, April 14, 2008.

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The atomic coordinates and structure factors (codes 2vps, 2vpm, and 2vob) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the Biotechnology and Biological Sciences Research Council (Structural Proteomics of Rational Targets) United Kingdom and Wellcome Trust Grants 082596, 079838, and 083481. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S7.

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¹ To whom correspondence should be addressed. Tel.: 44-1382-385745; Fax: 44-1382-345764; E-mail: w.n.hunter{at}dundee.ac.uk.

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