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J. Biol. Chem., Vol. 281, Issue 50, 38150-38158, December 15, 2006

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Biochemical and Genetic Analysis of Methylenetetrahydrofolate Reductase in Leishmania Metabolism and Virulence^*

Tim J. Vickers, Giuseppe Orsomando, Rocío Díaz de la Garza, David A. Scott, Song O. Kang, Andrew D. Hanson, and Stephen M. Beverley¹

From the Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110 and the Department of Horticultural Sciences, University of Florida, Gainesville, Florida 32611

Methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20 [EC] ) is the sole enzyme responsible for generation of 5-methyltetrahydrofolate, which is required for methionine synthesis and provision of methyl groups via S-adenosylmethionine. Genome analysis showed that Leishmania species, unlike Trypanosoma brucei and Trypanosoma cruzi, contain genes encoding MTHFR and two distinct methionine synthases. Leishmania MTHFR differed from those in other eukaryotes by the absence of a C-terminal regulatory domain. L. major MTHFR was expressed in yeast and recombinant enzyme was produced in Escherichia coli. MTHFR was not inhibited by S-adenosylmethionine and, uniquely among folate-metabolizing enzymes, showed dual-cofactor specificity with NADH and NADPH under physiological conditions. MTHFR null mutants (mthfr^–) lacked 5-methyltetrahydrofolate, the most abundant intracellular folate, and could not utilize exogenous homocysteine for growth. Under conditions of methionine limitation mthfr^– mutant cells grew poorly, whereas their growth was normal in standard culture media. Neither in vitro MTHFR activity nor the growth of mthfr^– mutants or MTHFR overexpressors were differentially affected by antifolates known to inhibit parasite growth via targets beyond dihydrofolate reductase and pteridine reductase 1. In a mouse model of infection mthfr^– mutants showed good infectivity and virulence, indicating that sufficient methionine is available within the parasitophorous vacuole to meet the needs of the parasite.

Received for publication, August 31, 2006 , and in revised form, October 10, 2006.

^* This work was supported by National Institutes of Health Grants AI 21903 (to S. M. B.) and R01 GM071382 (to A. D. H.) and European Molecular Biology Organization long term fellowship ALTF 106–2005 (to T. V.). 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 methods, Table S1, and Figs. S1–S4.

¹ To whom correspondence should be addressed: Box 8230, WA University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. E-mail: beverley{at}borcim.wustl.edu.

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