Expression and characterization of Rv0447c product, potentially the methyltransferase involved in tuberculostearic acid biosynthesis in Mycobacterium tuberculosis

Abstract

In this study, a previously uncharacterized gene (Rv0447c) of Mycobacterium tuberculosis, designated as an unknown fatty-acid methyltransferase (ufaA1), was cloned, expressed in Escherichia coli, and purified. The biochemical characterization of the purified protein (UfaA1) showed it to be a methyltransferase that catalyzes biosynthesis of the tuberculostearic acid (10-methylstearic-acid, TSA), a significant constituent lipid of the mycobacterial cell wall and a clinical marker of the disease. Here, we show that UfaA1 transfers the methyl group from S-adenosyl-l-methionine (SAM) to the double bond of oleic acid in phosphatidylethanolamine or phosphatidylcholine to produce TSA. Optimal activity was obtained between pH 7.0 and pH 8.0. The methyltransferase activity of UfaA1 was severely inhibited by S-adenosyl-l-homocysteine. The Km values for dioleyl phosphatidylethanolamine, SAM, and nicotinamide adenine dinucleotide phosphate were 14, 13, and 83 µM, respectively, with Vmax of 1.3-1.6 nmol/Min. These results identify the Rv0447c gene product of M. tuberculosis as the methyltransferase that catalyzes the biosynthesis of TSA. This provides new information in mycobacterial cell wall synthesis.

Expression of Genes for a Flavin Adenine Dinucleotide-Binding Oxidoreductase and a Methyltransferase from Mycobacterium chlorophenolicum Is Necessary for Biosynthesis of 10-Methyl Stearic Acid from Oleic Acid in Escherichia coli

“…In this study, 18:1Δ9 connected with phosphatidylcholine (PC) was 3 H-labeled by incubation with the recombinant UmaA protein, S -adenosyl-[methyl 3 H] methionine ( 3 H-labeled SAM), and NADPH. Additionally, Meena and Kolattukudy (2013) also demonstrated the synthesis of 19:0Me10 by a recombinant protein encoded by the ufaA1 gene from M. tuberculosis H 37 Rv. In this study, the methylester of 19:0Me10 was detected by gas chromatography (GC) with a flame-ionization detector in a mixture comprising a crude extract from E. coli cells expressing UfaA1, PC or phosphatidylethanolamine containing 18:1Δ9, SAM, and NADPH.…”

Tuberculostearic acid (TSA)-containing phosphatidylinositols as reliable marker to determineMycobacterium tuberculosisbacterial burden

“…The enzymatic transformation of FA 18:1 (OA) to FA 19:0 (TSA) has been controversial discussed and two mechanisms have been proposed. Initially thought to be solely associated with Rv0447c, a two-step mechanism was suggested for M. chlorophenolicum, putatively catalyzed by proteins WP_048472120 and WP_048472121, orthologues of WP_003917236 and WP_003420415 (corresponding to Rv3719 and Rv3720 of H37Rv) (28,29). We are convinced that further detailed analyses of the enzymatic transformation of FA 18:1 (OA) to FA 19:0 (TSA) in appropriate model systems will support studies on Mtb metabolism with regard to replication, dormancy, persistence and also reactivation.…”

The influence of AccD5 on AccD6 carboxyltransferase essentiality in pathogenic and non-pathogenic Mycobacterium

“…Given the high demand for propionyl-CoA carboxylase activity in Mtb pathogenesis, AccD5 protein in this bacterium may have undergone a strict evolutionary specialization towards the utilization of propionyl-CoA as the sole substrate of the reaction catalyzed. In Msm , however, the synthesis of the most representative methyl-branched tuberculostearic acid (10-methylstearic) can be considered “AccD5-independent” because the methyl moiety is derived from the S-methyl group of methionine in this case 33 34 . Thus, it can be expected that the requirement for propionyl-CoA carboxylase activity in Msm may be considerably lower than that in pathogenic species and that the substrate specificity of AccD5 in this bacterium may evolve towards acetyl-CoA utilization.…”