Publications

• Structures of Mycobacterium tuberculosis FadD10 protein reveal a new type of adenylate-forming enzyme.

  Liu Z., Ioerger T.R., Wang F., Sacchettini J.C.

  Mycobacterium tuberculosis has a group of 34 FadD proteins that belong to the adenylate-forming superfamily. They are classified as either fatty acyl-AMP ligases (FAALs) or fatty acyl-CoA ligases based on sequence analysis. FadD10, involved in the synthesis of a virulence-related lipopeptide, was ... >> More

  Mycobacterium tuberculosis has a group of 34 FadD proteins that belong to the adenylate-forming superfamily. They are classified as either fatty acyl-AMP ligases (FAALs) or fatty acyl-CoA ligases based on sequence analysis. FadD10, involved in the synthesis of a virulence-related lipopeptide, was mis-annotated as a fatty acyl-CoA ligase; however, it is in fact a FAAL that transfers fatty acids to an acyl carrier protein (Rv0100). In this study, we have determined the structures of FadD10 in both the apo-form and the complexed form with dodecanoyl-AMP, where we see for the first time an adenylate-forming enzyme that does not adopt a closed conformation for catalysis. Indeed, this novel conformation of FadD10, facilitated by its unique inter-domain and intermolecular interactions, is critical for the enzyme to carry out the acyl transfer onto Rv0100 rather than coenzyme A. This contradicts the existing model of FAALs that rely on an insertion motif for the acyltransferase specificity and thus makes FadD10 a new type of FAAL. We have also characterized the fatty acid preference of FadD10 through biological and structural analyses, and the data indicate long chain saturated fatty acids as the biological substrates of the enzyme. << Less

  J. Biol. Chem. 288:18473-18483(2013) [PubMed] [EuropePMC]

  This publication is cited by 8 other entries.

• The dual function of the Mycobacterium tuberculosis FadD32 required for mycolic acid biosynthesis.

  Leger M., Gavalda S., Guillet V., van der Rest B., Slama N., Montrozier H., Mourey L., Quemard A., Daffe M., Marrakchi H.

  Mycolic acids are major and specific lipids of Mycobacterium tuberculosis cell envelope. Their synthesis requires the condensation by Pks13 of a C(22)-C(26) fatty acid with the C(50)-C(60) meromycolic acid activated by FadD32, a fatty acyl-AMP ligase essential for mycobacterial growth. A combinati ... >> More

  Mycolic acids are major and specific lipids of Mycobacterium tuberculosis cell envelope. Their synthesis requires the condensation by Pks13 of a C(22)-C(26) fatty acid with the C(50)-C(60) meromycolic acid activated by FadD32, a fatty acyl-AMP ligase essential for mycobacterial growth. A combination of biochemical and enzymatic approaches demonstrated that FadD32 exhibits substrate specificity for relatively long-chain fatty acids. More importantly, FadD32 catalyzes the transfer of the synthesized acyl-adenylate onto specific thioester acceptors, thus revealing the protein acyl-ACP ligase function. Therefore, FadD32 might be the prototype of a group of M. tuberculosis polyketide-synthase-associated adenylation enzymes possessing such activity. A substrate analog of FadD32 inhibited not only the enzyme activity but also mycolic acid synthesis and mycobacterial growth, opening an avenue for the development of novel antimycobacterial agents. << Less

  Chem. Biol. 16:510-519(2009) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Mechanistic and functional insights into fatty acid activation in Mycobacterium tuberculosis.

  Arora P., Goyal A., Natarajan V.T., Rajakumara E., Verma P., Gupta R., Yousuf M., Trivedi O.A., Mohanty D., Tyagi A., Sankaranarayanan R., Gokhale R.S.

  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). Prese ... >> More

  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. << Less

  Nat. Chem. Biol. 5:166-173(2009) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Nonprocessive [2 + 2]e- off-loading reductase domains from mycobacterial nonribosomal peptide synthetases.

  Chhabra A., Haque A.S., Pal R.K., Goyal A., Rai R., Joshi S., Panjikar S., Pasha S., Sankaranarayanan R., Gokhale R.S.

  In mycobacteria, polyketide synthases and nonribosomal peptide synthetases (NRPSs) produce complex lipidic metabolites by using a thio-template mechanism of catalysis. In this study, we demonstrate that off-loading reductase (R) domain of mycobacterial NRPSs performs two consecutive [2 + 2]e(-) re ... >> More

  In mycobacteria, polyketide synthases and nonribosomal peptide synthetases (NRPSs) produce complex lipidic metabolites by using a thio-template mechanism of catalysis. In this study, we demonstrate that off-loading reductase (R) domain of mycobacterial NRPSs performs two consecutive [2 + 2]e(-) reductions to release thioester-bound lipopeptides as corresponding alcohols, using a nonprocessive mechanism of catalysis. The first crystal structure of an R domain from Mycobacterium tuberculosis NRPS provides strong support to this mechanistic model and suggests that the displacement of intermediate would be required for cofactor recycling. We show that 4e(-) reductases produce alcohols through a committed aldehyde intermediate, and the reduction of this intermediate is at least 10 times more efficient than the thioester-substrate. Structural and biochemical studies also provide evidence for the conformational changes associated with the reductive cycle. Further, we show that the large substrate-binding pocket with a hydrophobic platform accounts for the remarkable substrate promiscuity of these domains. Our studies present an elegant example of the recruitment of a canonical short-chain dehydrogenase/reductase family member as an off-loading domain in the context of assembly-line enzymology. << Less

  Proc. Natl. Acad. Sci. U.S.A. 109:5681-5686(2012) [PubMed] [EuropePMC]

  This publication is cited by 11 other entries.

• Versatile polyketide enzymatic machinery for the biosynthesis of complex mycobacterial lipids.

  Gokhale R.S., Saxena P., Chopra T., Mohanty D.

  The cell envelope of Mycobacterium tuberculosis (Mtb) is a treasure house of a variety of biologically active molecules with fascinating architectures. The decoding of the genetic blueprint of Mtb in recent years has provided the impetus for dissecting the metabolic pathways involved in the biosyn ... >> More

  The cell envelope of Mycobacterium tuberculosis (Mtb) is a treasure house of a variety of biologically active molecules with fascinating architectures. The decoding of the genetic blueprint of Mtb in recent years has provided the impetus for dissecting the metabolic pathways involved in the biosynthesis of lipidic metabolites. The focus of the Highlight is to emphasize the functional role of polyketide synthase (PKS) proteins in the biosynthesis of complex mycobacterial lipids. The catalytic as well as mechanistic versatility of PKS. in generating metabolic diversity and the significance of recently discovered fatty acyl-AMP ligases in establishing "biochemical crosstalk" between fatty acid synthases (FASs) and PKSs is described. The phenotypic heterogeneity and remodeling of the mycobacterial cell wall in its aetiopathogenesis is discussed. << Less

  Nat. Prod. Rep. 24:267-277(2007) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Selection of transposon mutants of Mycobacterium tuberculosis with increased macrophage infectivity identifies fadD23 to be involved in sulfolipid production and association with macrophages.

  Lynett J., Stokes R.W.

  Alterations to the composition or architecture of the mycobacterial cell envelope can affect the macrophage infectivity of the bacillus. To further characterize the mycobacterial gene products that modulate the interaction with host cells, we employed transposon mutagenesis and screened for mutant ... >> More

  Alterations to the composition or architecture of the mycobacterial cell envelope can affect the macrophage infectivity of the bacillus. To further characterize the mycobacterial gene products that modulate the interaction with host cells, we employed transposon mutagenesis and screened for mutants that demonstrated an enhanced binding affinity toward macrophages. After successive rounds of mutant selection and enrichment, a total of five mutants were isolated that harboured gene disruptions within loci involved in lipid synthetic pathways as well as genes coding for putative hypothetical proteins. One mutant in particular, with a disruption in the Rv3826 gene (fadD23), was repeatedly isolated during library screening. Analysis of the cell envelope constituents of the Tn : : fadD23 strain revealed a lack of sulfolipid production which was restored following complementation with the wild-type gene. << Less

  Microbiology 153:3133-3140(2007) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.