Publications

• Crystallization and preliminary X-ray crystallographic studies of the N-terminal domain of FadD28, a fatty-acyl AMP ligase from Mycobacterium tuberculosis.

  Goyal A., Yousuf M., Rajakumara E., Arora P., Gokhale R.S., Sankaranarayanan R.

  FadD28 from Mycobacterium tuberculosis belongs to the fatty-acyl AMP ligase (FAAL) family of proteins. It is essential for the biosynthesis of a virulent phthiocerol dimycocerosate (PDIM) lipid that is only found in the cell wall of pathogenic mycobacteria. The N-terminal domain, comprising of the ... >> More

  FadD28 from Mycobacterium tuberculosis belongs to the fatty-acyl AMP ligase (FAAL) family of proteins. It is essential for the biosynthesis of a virulent phthiocerol dimycocerosate (PDIM) lipid that is only found in the cell wall of pathogenic mycobacteria. The N-terminal domain, comprising of the first 460 residues, was crystallized by the hanging-drop vapour-diffusion method at 295 K. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.97, b = 60.74, c = 136.54 angstroms. The crystal structure of the N-terminal domain of FadD28 at 2.35 angstroms resolution has been solved using the MAD method. << Less

  Acta Crystallogr. F 62:350-352(2006) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Biosynthesis of cell envelope-associated phenolic glycolipids in Mycobacterium marinum.

  Vergnolle O., Chavadi S.S., Edupuganti U.R., Mohandas P., Chan C., Zeng J., Kopylov M., Angelo N.G., Warren J.D., Soll C.E., Quadri L.E.

  Phenolic glycolipids (PGLs) are polyketide synthase-derived glycolipids unique to pathogenic mycobacteria. PGLs are found in several clinically relevant species, including various Mycobacterium tuberculosis strains, Mycobacterium leprae, and several nontuberculous mycobacterial pathogens, such as ... >> More

  Phenolic glycolipids (PGLs) are polyketide synthase-derived glycolipids unique to pathogenic mycobacteria. PGLs are found in several clinically relevant species, including various Mycobacterium tuberculosis strains, Mycobacterium leprae, and several nontuberculous mycobacterial pathogens, such as M. marinum. Multiple lines of investigation implicate PGLs in virulence, thus underscoring the relevance of a deep understanding of PGL biosynthesis. We report mutational and biochemical studies that interrogate the mechanism by which PGL biosynthetic intermediates (p-hydroxyphenylalkanoates) synthesized by the iterative polyketide synthase Pks15/1 are transferred to the noniterative polyketide synthase PpsA for acyl chain extension in M. marinum. Our findings support a model in which the transfer of the intermediates is dependent on a p-hydroxyphenylalkanoyl-AMP ligase (FadD29) acting as an intermediary between the iterative and the noniterative synthase systems. Our results also establish the p-hydroxyphenylalkanoate extension ability of PpsA, the first-acting enzyme of a multisubunit noniterative polyketide synthase system. Notably, this noniterative system is also loaded with fatty acids by a specific fatty acyl-AMP ligase (FadD26) for biosynthesis of phthiocerol dimycocerosates (PDIMs), which are nonglycosylated lipids structurally related to PGLs. To our knowledge, the partially overlapping PGL and PDIM biosynthetic pathways provide the first example of two distinct, pathway-dedicated acyl-AMP ligases loading the same type I polyketide synthase system with two alternate starter units to produce two structurally different families of metabolites. The studies reported here advance our understanding of the biosynthesis of an important group of mycobacterial glycolipids. << Less

  J. Bacteriol. 197:1040-1050(2015) [PubMed] [EuropePMC]

  This publication is cited by 19 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.

• Open reading frame 3, which is adjacent to the mycocerosic acid synthase gene, is expressed as an acyl coenzyme A synthase in Mycobacterium bovis BCG.

  Fitzmaurice A.M., Kolattukudy P.E.

  The aim of this study was to test for expression of a 900-bp open reading frame (ORF), ORF3, located at the 5' end of the mycocerosic acid synthase gene in Mycobacterium bovis BCG and to determine the nature of the ORF3 protein. ORF3 was expressed as a 61-kDa C-terminal fusion protein with glutath ... >> More

  The aim of this study was to test for expression of a 900-bp open reading frame (ORF), ORF3, located at the 5' end of the mycocerosic acid synthase gene in Mycobacterium bovis BCG and to determine the nature of the ORF3 protein. ORF3 was expressed as a 61-kDa C-terminal fusion protein with glutathione S-transferase in Escherichia coli. Polyclonal rabbit antiserum, prepared against this fusion protein, cross-reacted with a 65-kDa protein in M. bovis BCG crude extracts. Since this protein was larger than that predicted from the nucleotide sequence (32 kDa), ORF3 was resequenced, revealing an ORF of 1,749 bp that encodes a 64.8-kDa protein containing 583 amino acids. Reverse transcription-PCR revealed that ORF3 is expressed in M. bovis BCG. The ORF3 product has a high degree of similarity to the acyladenylate family of enzymes. Immunoaffinity absorption chromatography was used to isolate the 65-kDa cross-reacting protein from M. bovis BCG. This purified protein catalyzed coenzyme A (CoA) ester synthesis of n-C10 to n-C18 fatty acids but not mycocerosic acids. ORF3 antibodies severely inhibited acyl-CoA synthase activities of the purified protein and extracts of M. bovis BCG, Mycobacterium smegmatis, and E. coli. They also showed immunological cross-reactivity with proteins in these extracts. Both the ORF3 protein and the acyl-CoA synthase activity were located in the cell cytosol or were loosely associated with the cell membrane. These results indicate that ORF3 encodes an acyl-CoA synthase-like protein. << Less

  J Bacteriol 179:2608-2615(1997) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Expanding the chemical diversity of natural esters by engineering a polyketide-derived pathway into Escherichia coli.

  Menendez-Bravo S., Comba S., Sabatini M., Arabolaza A., Gramajo H.

  Microbial fatty acid (FA)-derived molecules have emerged as promising alternatives to petroleum-based chemicals for reducing dependence on fossil hydrocarbons. However, native FA biosynthetic pathways often yield limited structural diversity, and therefore restricted physicochemical properties, of ... >> More

  Microbial fatty acid (FA)-derived molecules have emerged as promising alternatives to petroleum-based chemicals for reducing dependence on fossil hydrocarbons. However, native FA biosynthetic pathways often yield limited structural diversity, and therefore restricted physicochemical properties, of the end products by providing only a limited variety of usually linear hydrocarbons. Here we have engineered into Escherichia coli a mycocerosic polyketide synthase-based biosynthetic pathway from Mycobacterium tuberculosis and redefined its biological role towards the production of multi-methyl-branched-esters (MBEs) with novel chemical structures. Expression of FadD28, Mas and PapA5 enzymes enabled the biosynthesis of multi-methyl-branched-FA and their further esterification to an alcohol. The high substrate tolerance of these enzymes towards different FA and alcohol moieties resulted in the biosynthesis of a broad range of MBE. Further metabolic engineering of the MBE producer strain coupled this system to long-chain-alcohol biosynthetic pathways resulting in de novo production of branched wax esters following addition of only propionate. << Less

  Metab. Eng. 24:97-106(2014) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.