Mycolic Acid Biosynthesis Research Articles

S-Enantiomer of the Antitubercular Compound S006-830 Complements Activity of Frontline TB Drugs and Targets Biogenesis of Mycobacterium tuberculosis Cell Envelope.

A synthetic molecule S006-830, belonging to the class of thiophene-containing trisubstituted methanes, had shown good in vitro and in vivo bactericidal activity against drug-sensitive and drug-resistant Mycobacterium tuberculosis (Mtb). The molecule had also shown good druglike pharmacokinetic properties. However, S006-830 is a racemic mixture of two enantiomers, one of which could possess a better pharmacological profile than the other. We purified both the enantiomers on a chiral column and observed that S-enantiomer has a significantly higher inhibitory and cidal activity against Mtb than the R-enantiomer. Action of S-S006-830 was “synergistic” for rifampicin and “additive” for isoniazid and ethambutol. The combination of S-S006-830 and rifampicin produced 100% kill of Mtb within 8 days. In a chemical proteomics approach using matrix-bound compound to pull down its target protein(s) from Mtb membrane, FabG4 (β-ketoacyl CoA reductase, EC 1.1.1.100) emerged as the most likely target for S-S006-830. In target validation assays, the compound exhibited 2-fold higher inhibitory concentration for an Mtb construct overexpressing FabG4. In addition, it inhibited mycolic acid biosynthesis and formation of biofilms by Mtb. Molecular docking of S-S006-830 with FabG4 was consistent with the experimental data. These results support the development of S-S006-830 as a novel lead against tuberculosis.

A fluorescence-based reporter for monitoring expression of mycobacterial cytochrome bd in response to antibacterials and during infection

Cytochrome bd is a component of the oxidative phosphorylation pathway in many Gram-positive and Gram-negative bacteria. Next to its role as a terminal oxidase in the respiratory chain this enzyme plays an important role as a survival factor in the bacterial stress response. In Mycobacterium tuberculosis and related mycobacterial strains, cytochrome bd is an important component of the defense system against antibacterial drugs. In this report we describe and evaluate an mCherry-based fluorescent reporter for detection of cytochrome bd expression in Mycobacterium marinum. Cytochrome bd was induced by mycolic acid biosynthesis inhibitors such as isoniazid and most prominently by drugs targeting oxidative phosphorylation. We observed no induction by inhibitors of protein-, DNA- or RNA-synthesis. The constructed expression reporter was suitable for monitoring mycobacterial cytochrome bd expression during mouse macrophage infection and in a zebrafish embryo infection model when using Mycobacterium marinum. Interestingly, in both these infection models cytochrome bd levels were considerably higher than during in vitro culturing of M. marinum. The expression reporter described here can be a valuable tool for elucidating the role of cytochrome bd as a survival factor.

In Vitro Effect of DFC-2 on Mycolic Acid Biosynthesis in Mycobacterium tuberculosis.

DFC-2, a methyl 5-[2-(dimethylamino)ethoxy]-7,12-dioxo-7,12-dihydrodinaphtho[1,2-b:2',3'-d]furan-6-carboxylate, is reported to have antitubercular effects against Mycobacterium tuberculosis. At concentrations ranging from 0.19 to 0.39 μg/ml, DFC-2 inhibited both drug-susceptible and -resistant strains of M. tuberculosis. Microarray analyses were employed to gain insights into the molecular mechanisms of DFC-2's action in M. tuberculosis. The most affected functional gene category was "lipid biosynthesis," which is involved in mycolic acid synthesis. The decrease in transcription of genes related to mycolic acid synthesis was confirmed by RT-PCR. Furthermore, we found that DFC-2 triggered a reduction in mycolic acid levels, showing a similar pattern to that of mycolic acid synthesis inhibitor isoniazid. These results may explain how this compound kills mycobacteria efficiently by inhibiting mycolic acid synthesis.

In Silico Drug Design of Thiolactomycin Derivatives Against Mtb-KasA Enzyme to Inhibit Multidrug Resistance of Mycobacterium tuberculosis.

Tuberculosis (TB) is a leading infectious disease which kills a huge number of people every year over a decade, caused by Mycobacterium tuberculosis. The conventional drugs in the market are no longer effective due to the increasing mycobacterial resistance to antibiotics. Hence, the need of finding efficient drugs to solve this multiple drug resistant factor is becoming an immediate issue. The first-line drugs in current practice for the treatment of TB emphasize on mycolic acid, which protects the bacteria from an immune response generated by the host. A key enzyme involved in this mycolic acid biosynthesis, M. tuberculosis beta-ketoacyl-ACP synthase A (MTB-KasA) is a prime candidate in this study. Thiolactomycin is a natural product inhibitor has shown good inhibitory activity against MTB-KasA. Hence, several thiolactomycin derivatives collected from the literature were taken for absorption, distribution, metabolism, excretion and toxicity prediction, molecular docking and molecular dynamics simulation studies with MTB-KasA. The in silico drug designing methods used in this study suggests that the thiolactomycin derivatives are having a better binding activity against MTB-KasA and among them the ligand C14 is identified as a promising lead molecule to inhibit multidrug resistance of tuberculosis by showing a long time binding activity.

Structural and mechanistic comparison of the Cyclopropane Mycolic Acid Synthases (CMAS) protein family of Mycobacterium tuberculosis

Tuberculosis (TB) is a chronic disease caused by the bacillus Mycobacterium tuberculosis(Mtb) and remains a leading cause of mortality worldwide. The bacteria has an external wall which protects it from being killed, and the enzymes involved in the biosynthesis of the cell wall components have been proposed as promising targets for future drug development efforts. Cyclopropane Mycolic Acid Synthases (CMAS) constitute a group of ten homologous enzymes which belong to the mycolic acid biosynthesis pathway. These enzymes have S-adenosyl-l-methionine (SAM) dependent methyltransferase activity with a peculiarity, each one of them has strong substrate selectivity and reaction specificity, being able to produce among other things cyclopropanes or methyl-alcohol groups from the lipid olefin group. How each CMAS processes its substrate and how the specificity and selectivity are encoded in the protein sequence and structure, is still unclear.In this work, by using a combination of modeling tools, including comparative modeling, docking, all-atom MD and QM/MM methodologies we studied in detail the reaction mechanism of cmaA2, mmaA4, and mmaA1 CMAS and described the molecular determinants that lead to different products. We have modeled the protein-substrate complex structure and determined the free energy pathway for the reaction. The combination of modeling tools at different levels of complexity allows having a complete picture of the CMAS structure-activity relationship.

2-aminoimidazoles potentiate ß-lactam antimicrobial activity against Mycobacterium tuberculosis by reducing ß-lactamase secretion and increasing cell envelope permeability.

There is an urgent need to develop new drug treatment strategies to control the global spread of drug-sensitive and multidrug-resistant Mycobacterium tuberculosis (M. tuberculosis). The ß-lactam class of antibiotics is among the safest and most widely prescribed antibiotics, but they are not effective against M. tuberculosis due to intrinsic resistance. This study shows that 2-aminoimidazole (2-AI)-based small molecules potentiate ß-lactam antibiotics against M. tuberculosis. Active 2-AI compounds significantly reduced the minimal inhibitory and bactericidal concentrations of ß-lactams by increasing M. tuberculosis cell envelope permeability and decreasing protein secretion including ß-lactamase. Metabolic labeling and transcriptional profiling experiments revealed that 2-AI compounds impair mycolic acid biosynthesis, export and linkage to the mycobacterial envelope, counteracting an important defense mechanism reducing permeability to external agents. Additionally, other important constituents of the M. tuberculosis outer membrane including sulfolipid-1 and polyacyltrehalose were also less abundant in 2-AI treated bacilli. As a consequence of 2-AI treatment, M. tuberculosis displayed increased sensitivity to SDS, increased permeability to nucleic acid staining dyes, and rapid binding of cell wall targeting antibiotics. Transcriptional profiling analysis further confirmed that 2-AI induces transcriptional regulators associated with cell envelope stress. 2-AI based small molecules potentiate the antimicrobial activity of ß-lactams by a mechanism that is distinct from specific inhibitors of ß-lactamase activity and therefore may have value as an adjunctive anti-TB treatment.

Role of long-chain acyl-CoAs in the regulation of mycolic acid biosynthesis in mycobacteria.

One of the dominant features of the biology of Mycobacterium tuberculosis, and other mycobacteria, is the mycobacterial cell envelope with its exceptional complex composition. Mycolic acids are major and very specific components of the cell envelope and play a key role in its architecture and impermeability. Biosynthesis of mycolic acid (MA) precursors requires two types of fatty acid synthases, FAS I and FAS II, which should work in concert in order to keep lipid homeostasis tightly regulated. Both FAS systems are regulated at their transcriptional level by specific regulatory proteins. FasR regulates components of the FAS I system, whereas MabR and FadR regulate components of the FAS II system. In this article, by constructing a tight mabR conditional mutant in Mycobacterium smegmatis mc2155, we demonstrated that sub-physiological levels of MabR lead to a downregulation of the fasII genes, inferring that this protein is a transcriptional activator of the FAS II system. In vivo labelling experiments and lipidomic studies carried out in the wild-type and the mabR conditional mutant demonstrated that under conditions of reduced levels of MabR, there is a clear inhibition of biosynthesis of MAs, with a concomitant change in their relative composition, and of other MA-containing molecules. These studies also demonstrated a change in the phospholipid composition of the membrane of the mutant strain, with a significant increase of phosphatidylinositol. Gel shift assays carried out with MabR and PfasII as a probe in the presence of different chain-length acyl-CoAs strongly suggest that molecules longer than C18 can be sensed by MabR to modulate its affinity for the operator sequences that it recognizes, and in that way switch on or off the MabR-dependent promoter. Finally, we demonstrated the direct role of MabR in the upregulation of the fasII operon genes after isoniazid treatment.

Monitoring global protein thiol-oxidation and protein S-mycothiolation in Mycobacterium smegmatis under hypochlorite stress

Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes. Here, we used shotgun proteomics, OxICAT and RNA-seq transcriptomics to analyse protein S-mycothiolation, reversible thiol-oxidations and their impact on gene expression in Mycobacterium smegmatis under hypochlorite stress. In total, 58 S-mycothiolated proteins were identified under NaOCl stress that are involved in energy metabolism, fatty acid and mycolic acid biosynthesis, protein translation, redox regulation and detoxification. Protein S-mycothiolation was accompanied by MSH depletion in the thiol-metabolome. Quantification of the redox state of 1098 Cys residues using OxICAT revealed that 381 Cys residues (33.6%) showed >10% increased oxidations under NaOCl stress, which overlapped with 40 S-mycothiolated Cys-peptides. The absence of MSH resulted in a higher basal oxidation level of 338 Cys residues (41.1%). The RseA and RshA anti-sigma factors and the Zur and NrdR repressors were identified as NaOCl-sensitive proteins and their oxidation resulted in an up-regulation of the SigH, SigE, Zur and NrdR regulons in the RNA-seq transcriptome. In conclusion, we show here that NaOCl stress causes widespread thiol-oxidation including protein S-mycothiolation resulting in induction of antioxidant defense mechanisms in M. smegmatis. Our results further reveal that MSH is important to maintain the reduced state of protein thiols.

Essentiality of mmpL3 and impact of its silencing on Mycobacterium tuberculosis gene expression

MmpL3 is an inner membrane transporter of Mycobacterium tuberculosis responsible for the export of trehalose momomycolate, a precursor of the mycobacterial outer membrane component trehalose dimycolate (TDM), as well as mycolic acids bound to arabinogalactan. MmpL3 represents an emerging target for tuberculosis therapy. In this paper, we describe the construction and characterization of an mmpL3 knockdown strain of M. tuberculosis. Downregulation of mmpL3 led to a stop in bacterial division and rapid cell death, preceded by the accumulation of TDM precursors. MmpL3 was also shown to be essential for growth in monocyte-derived human macrophages. Using RNA-seq we also found that MmpL3 depletion caused up-regulation of 47 genes and down-regulation of 23 genes (at least 3-fold change and false discovery rate ≤1%). Several genes related to osmoprotection and metal homeostasis were induced, while several genes related to energy production and mycolic acids biosynthesis were repressed suggesting that inability to synthesize a correct outer membrane leads to changes in cellular permeability and a metabolic downshift.

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

Malonyl-coenzyme A (CoA) is a crucial extender unit for the synthesis of mycolic and other fatty acids in mycobacteria, generated in a reaction catalyzed by acetyl-CoA carboxylase. We previously reported on the essentiality of accD6Mtb encoding the functional acetyl-CoA carboxylase subunit in Mycobacterium tuberculosis. Strikingly, the homologous gene in the fast-growing, non-pathogenic Mycobacterium smegmatis - (accD6Msm) appeared to be dispensable, and its deletion did not influence the cell lipid content. Herein, we demonstrate that, despite the difference in essentiality, accD6Msm and accD6Mtb encode proteins of convergent catalytic activity in vivo. To identify an alternative, AccD6-independent, malonyl-CoA synthesis pathway in M. smegmatis, a complex genetic approach combined with lipid analysis was applied to screen all five remaining carboxyltransferase genes (accD1-accD5) with respect to their involvement in mycolic acid biosynthesis and ability to utilize acetyl-CoA as the substrate for carboxylation. This approach revealed that AccD1Msm, AccD2Msm and AccD3Msm are not essential for mycolic acid biosynthesis. Furthermore, we confirmed in vivo the function of AccD4Msm as an essential, long-chain acyl-CoA carboxyltransferase, unable to carboxylate short-chain substrate. Finally, our comparative studies unambiguously demonstrated between-species difference in in vivo ability of AccD5 carboxyltransferase to utilize acetyl-CoA that influences AccD6 essentiality in pathogenic and non-pathogenic mycobacteria.

A conditional mutant of the fatty acid synthase unveils unexpected cross talks in mycobacterial lipid metabolism.

Unlike most bacteria, mycobacteria rely on the multi-domain enzyme eukaryote-like fatty acid synthase I (FAS I) to make fatty acids de novo. These metabolites are precursors of the biosynthesis of most of the lipids present both in the complex mycobacteria cell wall and in the storage lipids inside the cell. In order to study the role of the type I FAS system in Mycobacterium lipid metabolism in vivo, we constructed a conditional mutant in the fas-acpS operon of Mycobacterium smegmatis and analysed in detail the impact of reduced de novo fatty acid biosynthesis on the global architecture of the cell envelope. As expected, the mutant exhibited growth defect in the non-permissive condition that correlated well with the lower expression of fas-acpS and the concomitant reduction of FAS I, confirming that FAS I is essential for survival. The reduction observed in FAS I provoked an accumulation of its substrates, acetyl-CoA and malonyl-CoA, and a strong reduction of C12 to C18 acyl-CoAs, but not of long-chain acyl-CoAs (C19 to C24). The most intriguing result was the ability of the mutant to keep synthesizing mycolic acids when fatty acid biosynthesis was impaired. A detailed comparative lipidomic analysis showed that although reduced FAS I levels had a strong impact on fatty acid and phospholipid biosynthesis, mycolic acids were still being synthesized in the mutant, although with a different relative species distribution. However, when triacylglycerol degradation was inhibited, mycolic acid biosynthesis was significantly reduced, suggesting that storage lipids could be an intracellular reservoir of fatty acids for the biosynthesis of complex lipids in mycobacteria. Understanding the interaction between FAS I and the metabolic pathways that rely on FAS I products is a key step to better understand how lipid homeostasis is regulated in this microorganism and how this regulation could play a role during infection in pathogenic mycobacteria.

Synthesis, screening and docking analysis of hispolon analogs as potential antitubercular agents

A series of 20 hispolons/dihydrohispolons were synthesized and characterized by spectral data. These compounds were subjected to in vitro antitubercular activity screening against Mycobacterium tuberculosis (H37Rv) strain. The synthesized compounds showed varied antitubercular activity ranging from 100 to 1.6μg/mL. Among the screened compounds, four compounds (H1, H2, H3 and H15) have shown moderate activity with MIC 25μg/mL. Potent activities were observed for the dihydrohispolon derivative H14 (MIC 1.6μg/mL) followed by H13 (6.25μg/mL) and H17 (12.5μg/mL), H19 (3.125μg/ML). Docking simulations gave good insights on the possible interactions between the tested compounds and β-keto acyl synthase enzyme (mtbFabH). Drug-inhibitor combination studies showed no synergism with the drugs targeting mycolic acid biosynthesis (isoniazid, ethambutol and thiolactomycin, a specific inhibitor of KAS-B enzyme) but showed significant synergism with other drugs including rifampicin and ciprofloxacin ascertaining the drug target for hispolons as inhibition of mycolic acid biosynthesis, probably via mtbFabH.

Identification of a Desaturase Involved in Mycolic Acid Biosynthesis in Mycobacterium smegmatis.

Mycolic acids are unique long chain fatty acids found in the cell walls of mycobacteria including the tubercle bacillus, Mycobacterium tuberculosis. The introduction of double bonds in mycolic acids remains poorly understood, however, genes encoding two potential aerobic desaturases have been proposed to be involved in this process. Here we show that one of these genes, desA1, is essential for growth of the saprophytic Mycobacterium smegmatis. Depletion of desA1 in a M. smegmatis conditional mutant led to reduction of mycolic acid biosynthesis and loss of viability. The DesA1-depleted cells exhibited two other phenotypes: using 14[C]-labelling, we detected the accumulation of minor mycolic acid-related species that migrated faster in a silver TLC plate. Spiral Time of Flight Mass Spectroscopic analysis suggested the presence of species with sizes corresponding to what were likely monoenoic derivatives of α-mycolic acids. Additionally, conditional depletion led to the presence of free fatty acyl species of lengths ~C26-C48 in the lysing cells. Cell viability could be rescued in the conditional mutant by Mycobacterium tuberculosis desA1, highlighting the potential of desA1 as a new drug target in pathogenic mycobacteria.

A metabolomics investigation of the function of the ESX-1 gene cluster in mycobacteria

The ESX-1 gene cluster, encoding the Type-VII secretion (T7S) system and its virulence associated proteins, ESAT-6 and CFP-10, is thought to be responsible for the transport of extracellular proteins across the hydrophobic and highly impermeable, cell envelope of Mycobacterium, and is involved in virulence in Mycobacterium tuberculosis, the causative agent of tuberculosis. Using a GCxGC-TOFMS metabolomics approach, a M. smegmatis ESX-1 knock-out strain (ΔESX-1ms) was compared to that of the M. smegmatis wild-type parent strain, and the metabolite markers due to the presence or absence of the ESX-1 gene cluster were identified. A general increase in specific metabolites in the ΔESX-1ms, confirmed the roles previously described for ESX-1 in mycolic acid biosynthesis and cell wall integrity. However, a number of other metabolite markers identified indicates ESX-1 has an additional role the in cell envelope structure, altering the levels of antioxidants and energy metabolism. Furthermore, the metabolome profiles correlated with the metabolomic variation observed when comparing a hyper- and hypo-virulent Beijing strain of M. tuberculosis, suggesting that the pathways which modulate virulence in M. tuberculosis are also influenced by ESX-1, reaffirming the previously described association of ESX-1 with virulence and cell envelope biogenesis.

The Mycobacterium tuberculosis desaturase DesA1 (Rv0824c) is a Ca2+ binding protein

The hallmark feature of Mycobacterium tuberculosis (M.tb) the causative agent of human tuberculosis, is its complex lipid rich cell wall comprised primarily of mycolic acids, long chain fatty acids that play a key role in structural stability and permeability of the cell wall. In addition, they are involved in inhibiting phagosome-lysosome fusion and aid in granuloma formation during the pathogenic process. M.tb DesA1 is an essential acyl-acyl carrier protein desaturase predicted to catalyze the introduction of position specific double bonds during the biosynthesis of mycolic acids. This protein is one among three annotated desaturases (DesA1-3) in the M.tb genome but is unique in containing a βγ-crystallin Greek key signature motif, a well-characterized fold known to mediate Ca2+ binding in both prokaryotic and eukaryotic organisms. Using Isothermal Titration Calorimetry and 45CaCl2 overlay, we demonstrate that Ca2+ binds to DesA1. Spectroscopic measurements suggested that this binding induces changes in protein conformation but does not lead to significant alterations in the secondary structure of the protein, a feature common to several βγ-crystallins. An M. smegmatis strain over-expressing M.tb desA1 showed a Ca2+ dependent variation in surface phenotype, revealing a functional role for Ca2+in DesA1 activity. This study represents the first identification of a Ca2+ binding βγ-crystallin in M.tb, emphasizing the implicit role of Ca2+ in the pathogenesis of M.tb.

Ser/Thr Phosphorylation Regulates the Fatty Acyl-AMP Ligase Activity of FadD32, an Essential Enzyme in Mycolic Acid Biosynthesis

Mycolic acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is a well known source of antituberculous drug targets. Among the promising new targets in the pathway, FadD32 is an essential enzyme required for the activation of the long meromycolic chain of mycolic acids and is essential for mycobacterial growth. Following the in-depth biochemical, biophysical, and structural characterization of FadD32, we investigated its putative regulation via post-translational modifications. Comparison of the fatty acyl-AMP ligase activity between phosphorylated and dephosphorylated FadD32 isoforms showed that the native protein is phosphorylated by serine/threonine protein kinases and that this phosphorylation induced a significant loss of activity. Mass spectrometry analysis of the native protein confirmed the post-translational modifications and identified Thr-552 as the phosphosite. Phosphoablative and phosphomimetic FadD32 mutant proteins confirmed both the position and the importance of the modification and its correlation with the negative regulation of FadD32 activity. Investigation of the mycolic acid condensation reaction catalyzed by Pks13, involving FadD32 as a partner, showed that FadD32 phosphorylation also impacts the condensation activity. Altogether, our results bring to light FadD32 phosphorylation by serine/threonine protein kinases and its correlation with the enzyme-negative regulation, thus shedding a new horizon on the mycolic acid biosynthesis modulation and possible inhibition strategies for this promising drug target.

Attenuation of Mycobacterium species through direct and macrophage mediated pathway by unsymmetrical diaryl urea

Tuberculosis is a major threat for mankind and the emergence of resistance strain of Mycobacterium tuberculosis (Mtb) against first line antibiotics makes it lethal for human civilization. In this study, we have synthesized different diaryl urea derivatives targeting the inhibition of mycolic acid biosynthesis. Among the 39 synthesized molecules, compounds 46, 57, 58 and 86 showed MIC values ≤ 10 μg/ml against H37Rv and mc26030 strains. The best molecule with a methyl at ortho position of the first aromatic ring and prenyl group at the meta position of the second aromatic ring showed the MIC value of 5.2 μg/ml and 1 μg/ml against H37Rv and mc26030 respectively, with mammalian cytotoxicity of 163.4 μg/ml. The effective compounds showed selective inhibitory effect on mycolic acid (epoxy mycolate) biosynthesis in 14C-radiolabelled assay. At the same time these molecules also executed their potent immunomodulatory activity by up-regulation of IFN-γ and IL-12 and down-regulation of IL-10.

Evaluation of the inhibitory activity of (aza)isoindolinone-type compounds: toward in vitro InhA action, Mycobacterium tuberculosis growth and mycolic acid biosynthesis.

Inhibitors of the Mycobacterium tuberculosis enoyl-ACP reductase (InhA) are considered as potential promising therapeutics for the treatment of tuberculosis. Previously, we reported that azaisoindolinone-type compounds displayed, in vitro, inhibitory activity toward InhA. Herein, we describe chemical modifications of azaisoindolinone scaffold, the synthesis of 15 new compounds and their evaluations toward the in vitro InhA activity. Based on these results, a structure-InhA inhibitory activity relationship analysis and a molecular docking study, using the conformation of InhA found in the 2H7M crystal structure, were carried out to predict a possible mode of interaction of the best (aza)isoindolinone-type inhibitors with InhA in vitro. Then, the work was extended toward evaluations of these compounds against Mycobacterium tuberculosis (Mtb) growth, and finally, some of them were also investigated in respect of their ability to inhibit mycolic acid biosynthesis inside mycobacteria. Although, some azaisoindolinones were able to inhibit InhA activity and Mtb growth in vitro, they did not inhibit the mycolic acid biosynthesis inside Mtb.

Structure of the Essential Mtb FadD32 Enzyme: A Promising Drug Target for Treating Tuberculosis

Mycolic acids are indispensible lipids of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), and contribute to the distinctive architecture and impermeability of the mycobacterial cell envelope. FadD32 plays a pivotal role in mycolic acid biosynthesis by functionally linking fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic pathways. FadD32, a fatty acyl-AMP ligase (FAAL), represents one of the best genetically and chemically validated new TB drug targets. We have determined the three-dimensional crystal structure of Mtb FadD32 in complex with a ligand specifically designed to stabilize the catalytically active adenylate-conformation, which provides a foundation for structure-based drug design efforts against this essential protein. The structure also captures the unique interactions of a FAAL-specific insertion sequence and provides insight into the specificity and mechanism of fatty acid transfer.

Analysis of coenzyme A activated compounds in actinomycetes.

Acyl-CoAs are crucial compounds involved in essential metabolic pathways such as the Krebs cycle and lipid, carbohydrate, and amino acid metabolisms, and they are also key signal molecules involved in the transcriptional regulation of lipid biosynthesis in many organisms. In this study, we took advantage of the high selectivity of mass spectrometry and developed an ion-pairing reverse-phase high-pressure liquid chromatography electrospray ionization high-resolution mass spectrometry (IP-RP-HPLC/ESI-HRMS) method to carry on a comprehensive analytical determination of the wide range of fatty acyl-CoAs present in actinomycetes. The advantage of using a QTOF spectrometer resides in the excellent mass accuracy over a wide dynamic range and measurements of the true isotope pattern that can be used for molecular formula elucidation of unknown analytes. As a proof of concept, we used this assay to determine the composition of the fatty acyl-CoA pools in Mycobacterium, Streptomyces, and Corynebacterium species, revealing an extraordinary difference in fatty acyl-CoA amounts and species distribution between the three genera and between the two species of mycobacteria analyzed, including the presence of different chain-length carboxy-acyl-CoAs, key substrates of mycolic acid biosynthesis. The method was also used to analyze the impact of two fatty acid synthase inhibitors on the acyl-CoA profile of Mycobacterium smegmatis, which showed some unexpected low levels of C24 acyl-CoAs in the isoniazid-treated cells. This robust, sensitive, and reliable method should be broadly applicable in the studies of the wide range of bacteria metabolisms in which acyl-CoA molecules participate.