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J. Biol. Chem., Vol. 280, Issue 49, 40699-40706, December 9, 2005

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p-Hydroxybenzoic Acid Synthesis in Mycobacterium tuberculosis^*

Gustavo Stadthagen, Jana Korduláková, Ruth Griffin1, Patricia Constant¶, Iveta Bottová2, Nathalie Barilone, Brigitte Gicquel, Mamadou Daffé¶, and Mary Jackson3

From the Unité deGénétique Mycobactérienne, Institut Pasteur, 75015, Paris, France, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom, and ^¶Département Mécanismes Moléculaires des Infections Mycobactériennes, Institut de Pharmacologie et de Biologie structurale, CNRS, Toulouse 31077 cedex 04, France

Glycosylated p-hydroxybenzoic acid methyl esters and structurally related phenolphthiocerol glycolipids are important virulence factors of Mycobacterium tuberculosis. Although both types of molecules are thought to be derived from p-hydroxybenzoic acid, the origin of this putative biosynthetic precursor in mycobacteria remained to be established. We describe the characterization of a transposon mutant of M. tuberculosis deficient in the production of all forms of p-hydroxybenzoic acid derivatives. The transposon was found to be inserted in Rv2949c, a gene located in the vicinity of the polyketide synthase gene pks15/1, involved in the elongation of p-hydroxybenzoate to phenolphthiocerol in phenolic glycolipid-producing strains. A recombinant form of the Rv2949c enzyme was produced in the fast-growing non-pathogenic Mycobacterium smegmatis and purified to near homogeneity. The recombinant enzyme catalyzed the removal of the pyruvyl moiety of chorismate to form p-hydroxybenzoate with an apparent K_m value for chorismate of 19.7 µM and a k_cat value of 0.102 s^-1. Strong inhibition of the reaction by p-hydroxybenzoate but not by pyruvate was observed. These results establish Rv2949c as a chorismate pyruvate-lyase responsible for the direct conversion of chorismate to p-hydroxybenzoate and identify Rv2949c as the sole enzymatic source of p-hydroxybenzoic acid in M. tuberculosis.

Received for publication, July 29, 2005 , and in revised form, October 6, 2005.

^* This work was supported by the Institut Pasteur, the European Commission, within the 6th Framework Program contract number LSHP-CT-2003-503367, the Heiser Program for Research in Leprosy and Tuberculosis (to J. K.), the CONACyT program from Mexico (to G. S.), and the Marie Curie Training Site program number CT-2000-00058 from the European Commission (to I. B.). 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 Present address: Centre for Molecular Microbiology and Infection, Imperial College, London, United Kingdom.

² Present address: Dept. of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina CH-1, 84215 Bratislava, Slovak Republic.

³ To whom correspondence should be addressed: Unité deGénétique Mycobactérienne, Institut Pasteur, 25 rue du Dr. Roux, 75015, Paris, France. Tel.: 33-1-45-68-88-77; Fax: 33-1-45-68-88-43; E-mail: mjackson{at}pasteur.fr.

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