Mechanistic and functional insights into fatty acid activation in Mycobacterium tuberculosis

National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
Nature Chemical Biology (Impact Factor: 13). 01/2009; 5(3):166-173. DOI: 10.1038/nchembio.143


The recent discovery of fatty acyl-AMP ligases (FAALs) in Mycobacterium tuberculosis (Mtb) provided a new perspective of fatty acid activation. These proteins convert fatty acids to the corresponding adenylates, which are intermediates of acyl-CoA–synthesizing fatty acyl-CoA ligases (FACLs). Presently, it is not evident how obligate pathogens such as Mtb have evolved such new themes of functional versatility and whether the activation of fatty acids to acyladenylates could indeed be a general mechanism. Here, based on elucidation of the first structure of an FAAL protein and by generating loss-of-function and gain-of-function mutants that interconvert FAAL and FACL activities, we demonstrate that an insertion motif dictates formation of acyladenylate. Because FAALs in Mtb are crucial nodes in the biosynthetic network of virulent lipids, inhibitors directed against these proteins provide a unique multipronged approach to simultaneously disrupting several pathways.

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Available from: Pooja Arora, Jan 17, 2014
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    • "Despite the challenges mentioned above, several drug candidates are currently under development and have a good chance to enter the market. Promising approaches for drug development include targeting synthesis of lipids as nutrients [25], [26] and synthesis of mycolic acids as major components of the cell wall [27]. In the last decade, researchers have identified compounds that kill dormant bacteria by intracellular NO release, such as the bicyclic nitroimidazoles, PA-824 [28], and OPC-67683, as well as compounds that affect ATP-synthesis such as TMC207 [29] and nitrofuranylamide compounds with so far unknown mode of action [30]. "
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    ABSTRACT: Tuberculosis is considered to be one of the world's deadliest disease with 2 million deaths each year. The need for new antitubercular drugs is further exacerbated by the emergence of drug-resistance strains. Despite multiple recent efforts, the majority of the hits discovered by traditional target-based screening showed low efficiency in vivo. Therefore, there is heightened demand for whole-cell based approaches directly using host-pathogen systems. The phenotypic host-pathogen assay described here is based on the monitoring of GFP-expressing Mycobacterium marinum during infection of the amoeba Acanthamoeba castellanii. The assay showed straight-forward medium-throughput scalability, robustness and ease of manipulation, demonstrating its qualities as an efficient compound screening system. Validation with a series of known antitubercular compounds highlighted the advantages of the assay in comparison to previously published macrophage-Mycobacterium tuberculosis-based screening systems. Combination with secondary growth assays based on either GFP-expressing D. discoideum or M. marinum allowed us to further fine-tune compound characterization by distinguishing and quantifying growth inhibition, cytotoxic properties and antibiotic activities of the compounds. The simple and relatively low cost system described here is most suitable to detect anti-infective compounds, whether they present antibiotic activities or not, in which case they might exert anti-virulence or host defense boosting activities, both of which are largely overlooked by classical screening approaches.
    PLoS ONE 01/2014; 9(1):e87834. DOI:10.1371/journal.pone.0087834 · 3.23 Impact Factor
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    • "While fadD18 appears to be a truncated paralogue of fadD19, the three others encode full-length proteins sharing less than 30% amino acid sequence identity (Cole et al., 1998; Camus et al., 2002). FadD17 and FadD19 have been reported to catalyse the CoA thioesterification of longchain fatty acids (Trivedi et al., 2004; Arora et al., 2009). "
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    ABSTRACT: The cholesterol catabolic pathway occurs in most mycolic acid-containing actinobacteria, such as Rhodococcus jostii RHA1, and is critical for Mycobacterium tuberculosis (Mtb) during infection. FadD3 is one of four predicted acyl-CoA synthetases potentially involved in cholesterol catabolism. A ΔfadD3 mutant of RHA1 grew on cholesterol to half the yield of wild-type and accumulated 3aα-H-4α(3'-propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP), consistent with the catabolism of half the steroid molecule. This phenotype was rescued by fadD3 of Mtb. Moreover, RHA1 but not ΔfadD3 grew on HIP. Purified FadD3(Mtb) catalyzed the ATP-dependent CoA thioesterification of HIP and its hydroxylated analogs, 5α-OH HIP and 1β-OH HIP. The apparent specificity constant (k(cat) /K(m) ) of FadD3(Mtb) for HIP was 7.3 ± 0.3 × 10(5) M(-1) s(-1) , 165-times higher than for 5α-OH HIP, while the apparent K(m) for CoASH was 110 ± 10 μM. In contrast to enzymes involved in the catabolism of rings A and B, FadD3(Mtb) did not detectably transform a metabolite with a partially degraded C17 side chain. Overall, these results indicate that FadD3 is a HIP-CoA synthetase that initiates catabolism of steroid rings C and D after side chain degradation is complete. These findings are consistent with the actinobacterial kstR2 regulon encoding ring C/D degradation enzymes.
    Molecular Microbiology 11/2012; 87(2). DOI:10.1111/mmi.12095 · 4.42 Impact Factor
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    • "Since the enzymes form thioester derivatives through an acyl-adenylate intermediate, this superfamily is also known as "acyl-adenylate/thioester-forming" superfamily. Apart from FACL (Fatty Acyl CoA Ligases), which transfer the acyl-adenylate intermediate to CoA, recently a new family of enzymes called FAAL (Fatty Acyl AMP Ligases) which transfer acyl adenylate to carrier protein domains of adjacent NRPS/PKS clusters have been discovered in Mycobacterium tuberculosis [2,3]. "
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    ABSTRACT: Enzymes belonging to acyl:CoA synthetase (ACS) superfamily activate wide variety of substrates and play major role in increasing the structural and functional diversity of various secondary metabolites in microbes and plants. However, due to the large sequence divergence within the superfamily, it is difficult to predict their substrate preference by annotation transfer from the closest homolog. Therefore, a large number of ACS sequences present in public databases lack any functional annotation at the level of substrate specificity. Recently, several examples have been reported where the enzymes showing high sequence similarity to luciferases or coumarate:CoA ligases have been surprisingly found to activate fatty acyl substrates in experimental studies. In this work, we have investigated the relationship between the substrate specificity of ACS and their sequence/structural features, and developed a novel computational protocol for in silico assignment of substrate preference. We have used a knowledge-based approach which involves compilation of substrate specificity information for various experimentally characterized ACS and derivation of profile HMMs for each subfamily. These HMM profiles can accurately differentiate probable cognate substrates from non-cognate possibilities with high specificity (Sp) and sensitivity (Sn) (Sn = 0.91-1.0, Sp = 0.96-1.0) values. Using homologous crystal structures, we identified a limited number of contact residues crucial for substrate recognition i.e. specificity determining residues (SDRs). Patterns of SDRs from different subfamilies have been used to derive predictive rules for correlating them to substrate preference. The power of the SDR approach has been demonstrated by correct prediction of substrates for enzymes which show apparently anomalous substrate preference. Furthermore, molecular modeling of the substrates in the active site has been carried out to understand the structural basis of substrate selection. A web based prediction tool has been developed for automated functional classification of ACS enzymes. We have developed a novel computational protocol for predicting substrate preference for ACS superfamily of enzymes using a limited number of SDRs. Using this approach substrate preference can be assigned to a large number of ACS enzymes present in various genomes. It can potentially help in rational design of novel proteins with altered substrate specificities.
    BMC Bioinformatics 01/2010; 11(1):57. DOI:10.1186/1471-2105-11-57 · 2.58 Impact Factor
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