The mycolyltransferase 85A, a putative drug target of Mycobacterium tuberculosis: Development of a novel assay and quantification of glycolipid-status of the mycobacterial cell wall

Abstract

CitationThe mycolyltransferase 85A, a putative drug target of 55 56 57 58 59 60 61 62 63 64 65 66 67 68 The enzymes of the Antigen 85 complex (Ag85A, B, and C) possess mycolyltransferase activity and catalyze the synthesis of the most abundant glycolipid of the mycobacterial cell wall, the cord factor. The cord factor (trehalose 6,6'-dimycolate, TDM) is essential for the integrity of the mycobacterial cell wall and pathogenesis of the bacillus. Thus, TDM biosynthesis is regarded as a potential drug target for control of Mycobacterium tuberculosis infections. Trehalose 6,6'-dimycolate (TDM) is synthesized from two molecules of trehalose-6'-monomycolate (TMM) by antigen 85A. We report here a novel enzyme assay using the natural substrate TMM. The novel colorimetric assay is based on the quantification of glucose from the degradation of trehalose, which is the product from catalytic activity of antigen 85A.Using the new assay, K m and K cat were determined with values of 129.6 ± 8.1 µM and 65.4 ± 4.1 min -1 , respectively. This novel assay is also suitable for robust high-throughput screening (HTS) for compound library screening against mycolyltransferase (antigen 85A). The assay is significantly faster and more convenient to use than all assays currently in use. The assay has a very low coefficient of variance (0.04) in 96-well plates and shows a Z′ factor of 0.67-0.73, indicating the robustness of the assay. In addition, this new assay is highly suitable for the quantification of total TMM of the mycobacterial cell envelope.

Mycoloyltransferases: A large and major family of enzymes shaping the cell envelope of Corynebacteriales

“…In parallel, Elamin et al (54) developed a colorimetric assay with FbpA using the natural TMM substrate to form TDM and trehalose, the latter being quantified by a series of coupled reactions (conversion into glucose which is oxidized in gluconic acid and hydrogen peroxide ultimately used to form the oxidized (brown) form of o-Dianisidine). Later, an ESI-MS (ElectroSpray Ionisation-Mass Spectrometry) assay was reported that uses a labelfree approach and permits a complete dissection of the kinetic steps of the mycoloyl transfer from TMM to trehalose, TMM or other derivatives (55).…”

The Mycobacterial Cell Envelope--Lipids

“…4). High-throughput screening assays were reported for FadD32 (Galandrin et al 2013) and the antigens 85 Elamin et al 2009;Sanki et al 2009;Favrot et al 2013). The latter assays led to the identification of a number of inhibitors of antigens 85, one of them, known as ebselen, also shows activity against Mtb in culture (MIC of 20 mg/mL).…”

Antigen 85C Inhibition Restricts Mycobacterium tuberculosis Growth through Disruption of Cord Factor Biosynthesis

“…Phosphonate inhibitors of Ag85C have been synthesized, with the most active molecules possessing a MIC range of 188 to 319 g/ml against M. avium in broth culture, with optical density (OD) as readout (20). Recently, modified enzymatic assays for high-throughput screening of Ag85 proteins have been reported (12,19). However, Ag85 antagonists, which inhibit division of pathogenic M. tuberculosis, and their mechanisms of action have not yet been reported.…”