Figure 1.
Proposed pathways for the synthesis of hydroxy-, keto-, methoxy- and epoxymycolic acids in mycobacteria [7], [24].
A common intermediate for various R groups is used as the starting point. Where MmaA2 and CmaA2 are involved in the formation of cis cyclopropane group, CmaA2 and an yet to identified enzyme (indicated by?) catalyze trans-cyclopropanation [48], [67]. The details of the individual R groups are shown in Fig. S1. * indicates that it is not known whether the cyclopropanation step follows or precedes oxygenation. All protons (except for the isolated groups) that have been target for NMR data analysis have been shown in red. The OH group shown in italics and underlined in the box at the left corner of the figure was converted to a methoxy group during saponification of mycolic acids; the process generated mycolic acids methyl esters (MAMEs).
Figure 2.
Comparison of the primary structures of Rv0132c (fHMAD) and three Mer homologs, Fgd1, MkMer and Adf.
A ClustalW comparison was refined manually based on X-ray crystallographic structures of F420-dependent glucose-6-phosphate dehydrogenase (Fgd1) of Mycobacterium tuberculosis, F420-dependent methylenetetrahydromethanopterin reductase from Methanopyrus kandleri (MkMer), and F420-dependent secondary alcohol dehydrogenase (Adf) from Methanoculleus thermophilicus [32], [39], [40], [68]. Residue labels: yellow shade and underlined, determined F420-binding residue; yellow shade, predicted F420-intercating residue; turquoise shade, forming positively charged pocket for binding the phosphate of glucose-6-phopsphate in Fgd1; green shade, residue involved in binding a citrate ion [32].
Figure 3.
Thin layer chromatography (TLC) profiles of methyl esters of mycolic acids extracted from various Mycobacterium smegmatis strains grown in the absence and presence of PA-824.
Wild type (wt) and ΔfbiC strains of M. smegmatis carrying the indicated plasmids were analyzed (lane label, name of plasmid): None, no plasmid; pSMT3, pSMT3 (vector control); hma, pEP-hma; rv0132c, pEP-rv0132c; rv0132c/hma, pEP-rv0132c/hma. (+) and (-), cultivation of M. smegmatis (pEP-rv0132c/hma) with and without PA-824 (100 microgram per ml), respectively. Mycolic acid types: α, α′, epoxy (E), hydroxy (H), and keto (K) [Fig. S1 shows the respective chemical structures.]. Panel A: rv0132c causing the conversion of H-MAs to K-MAs in wild-type M. smegmatis; Panel B: Requirement of fbiC for the production of K-MAs in M. smegmatis (pEP-rv0132c/hma) [Note: The left most lane is for wt strain, used as control]. Panel C: Inhibition of the production of K-MAs in M. smegmatis (pEP-rv0132c/hma) by PA-824.
Figure 4.
MALDI-TOF mass spectra of methyl esters of hydroxymyoclic acids (A) and ketomycolic acids (B) recovered from engineered Mycobacterium smegmatis strains.
Hydroxymycolic acids were obtained from the lane hma and ketomycolic acids were from lane rv0132c/hma (Fig. 3A). Only a part of each spectrum is shown and the annotations for the ion masses are based on reference [23]: labels H & K, ions from hydroxy- and ketomycolic acids preparations; numbers 77–82: total number of carbon atoms in free acids. The unlabeled peaks belong to unidentified species that were present in both preparations.
Figure 5.
Proton NMR spectra of methyl esters of mycolic acids recovered from engineered Mycobacterium smegmatis strains.
The sources of hydroxymycolic acids or H-MAs (A) and Ketomycolic acids or K-MAs (B) were same as that indicated in the legend of Fig. 4. Epoxymycolic acids or E-MAs (C) were from lane “None” in Fig. 3. In each case the inset shows expansion of the relevant regions.