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J. Biol. Chem., Vol. 279, Issue 22, 23668-23678, May 28, 2004

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Arginase Plays a Pivotal Role in Polyamine Precursor Metabolism in Leishmania

CHARACTERIZATION OF GENE DELETION MUTANTS^*

Sigrid C. Roberts, Michael J. Tancer, Michelle R. Polinsky, K. Michael Gibson, Olle Heby¶, and Buddy Ullman||

From the Department of Biochemistry and Molecular Biology, the Department of Molecular and Medical Genetics, Biochemical Genetics Laboratory, Oregon Health & Science University, Portland, Oregon 97239-3098 and the ^¶Department of Molecular Biology, Umea University, Umea SE-901 87, Sweden

The polyamine pathway of protozoan parasites has been successfully targeted in anti-parasitic therapies and is significantly different from that of the mammalian host. To gain knowledge into the metabolic routes by which parasites synthesize polyamines and their precursors, the arginase gene was cloned from Leishmania mexicana, and arg null mutants were created by double targeted gene replacement and characterized. The ARG sequence exhibited significant homology to ARG proteins from other organisms and predicted a peroxisomal targeting signal (PTS-1) that steers proteins to the glycosome, an organelle unique to Leishmania and related parasites. ARG was subsequently demonstrated to be present in the glycosome, whereas the polyamine biosynthetic enzymes, in contrast, were shown to be cytosolic. The arg knockouts expressed no ARG activity, lacked an intracellular ornithine pool, and were auxotrophic for ornithine or polyamines. The ability of the arg null mutants to proliferate could be restored by pharmacological supplementation, either with low putrescine or high ornithine or spermidine concentrations, or by complementation with an arginase episome. Transfection of an arg construct lacking the PTS-1 directed the synthesis of an arg that mislocalized to the cytosol and notably also complemented the genetic lesion and restored polyamine prototrophy to the arg parasites. This molecular, biochemical, and genetic dissection of ARG function in L. mexicana promastigotes establishes: (i) that the enzyme is essential for parasite viability; (ii) that Leishmania, unlike mammalian cells, expresses only one ARG activity; (iii) that the sole vital function of ARG is to provide polyamine precursors for the parasite; and (iv) that ARG is present in the glycosome, but this subcellular milieu is not essential for its role in polyamine biosynthesis.

Received for publication, February 24, 2004 , and in revised form, March 4, 2004.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY386701.

^* This work was supported in part by Grants AI10096 (to S. C. R.) and AI41622 (to B. U.) from the NIAID, National Institutes of Health, The Swedish Research Council (to O. H.), the J. C. Kempe Memorial Foundation (to O. H.), and the Royal Physiographic Society in Lund (to O. H.). 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.

^|| To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, L224, Oregon Health & Science University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97239-3098. Tel.: 503-494-8437; Fax: 503-494-8393; E-mail: ullmanb{at}ohsu.edu.

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