P-Hydroxybenzoic Acid Synthesis in Mycobacterium tuberculosis

Gustavo Stadthagen,Jana Korduláková,Ruth Griffin,Patricia Constant,Iveta Bottová,Nathalie Barilone,Brigitte Gicquel,Mamadou Daffé,Mary Jackson

doi:10.1074/jbc.m508332200

Abstract

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 microm 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.

Highlights

The chorismate pathway, present only in bacteria, fungi, and plants, provides a wealth of compounds with diverse biological functions, including aromatic amino acids, folate cofactors, menaquinones, ubiquinones, pigments, and iron-chelating siderophores
Because of the important roles played by phenolic glycolipids (PGL) and p-HBADs in the pathogenesis of mycobacterial infections, their biosynthesis has stimulated some interest, and, during the last decade, several genes involved in the synthesis of the lipid core of PGL and in the methylation and glycosylation of PGL and p-HBADs have been described [3, 12,13,14,15,16,17,18,19,20,21,22]
Isolation of a Rv2949c Mutant of M. tuberculosis Mt103 from a Transposon Mutant Library—Mutants of M. tuberculosis Mt103 carrying transposon insertions in a region of the chromosome associated with the synthesis of PGL and structurally related glycosylated p-HBADs were recently isolated [10]

Summary

Introduction

The chorismate pathway, present only in bacteria, fungi, and plants, provides a wealth of compounds with diverse biological functions, including aromatic amino acids, folate cofactors, menaquinones, ubiquinones, pigments, and iron-chelating siderophores. We describe the characterization of a transposon mutant of M. tuberculosis deficient in the production of all forms of p-hydroxybenzoic acid derivatives.

Results

Conclusion