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J. Biol. Chem., Vol. 280, Issue 9, 8069-8078, March 4, 2005

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Functional Demonstration of Reverse Transsulfuration in the Mycobacterium tuberculosis Complex Reveals That Methionine Is the Preferred Sulfur Source for Pathogenic Mycobacteria^*

Paul R. Wheeler, Nicholas G. Coldham, Lisa Keating, Stephen V. Gordon, Esen E. Wooff, Tanya Parish¶, and R. Glyn Hewinson

From the Tuberculosis Research Group, Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone KT15 3NB, United Kingdom and the ^¶Centre for Infectious Diseases, Barts and the London, Queen Mary's School of Medicine and Dentistry, London E1 2AA, United Kingdom

Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex although it is not obvious from examination of the genome annotation how these bacteria utilize methionine. Given that genome annotation is a largely predictive process, key challenges are to validate these predictions and to fill in gaps for known functions for which genes have not been annotated. We have addressed these issues by functional analysis of methionine metabolism. Transport, followed by metabolism of ³⁵S methionine into the cysteine adduct mycothiol, demonstrated the conversion of exogenous methionine to cysteine. Mutational analysis and cloning of the Rv1079 gene showed it to encode the key enzyme required for this conversion, cystathionine -lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine -synthase (CGS), but demonstration of a -elimination reaction with cystathionine as well as the -replacement reaction yielding cystathionine showed it encodes a bifunctional CGL/CGS enzyme. Consistent with this, a Rv1079 mutant could not incorporate sulfur from methionine into cysteine, while a cysA mutant lacking sulfate transport and a methionine auxotroph was hypersensitive to the CGL inhibitor propargylglycine. Thus, reverse transsulfuration alone, without any sulfur recycling reactions, allows M. tuberculosis to use methionine as the sole sulfur source. Intracellular cysteine was undetectable so only the CGL reaction occurs in intact mycobacteria. Cysteine desulfhydrase, an activity we showed to be separable from CGL/CGS, may have a role in removing excess cysteine and could explain the ability of M. tuberculosis to recycle sulfur from cysteine, but not methionine.

Received for publication, November 5, 2004

^* This work was supported in part by the Environment and Rural Affairs (DEFRA). 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 Data.

To whom correspondence should be addressed: Tuberculosis Research Group, Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UK. Tel.: 44-0-1932-357506; Fax: 44-0-1932-357684; E-mail: p.wheeler{at}vla.defra.gsi.gov.uk.

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