Turnover of lipids in Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354

Dhariwal, Kuldeep R.; Chander, Avinash; Venkitasubramanian, T. A.

doi:10.1007/bf00408735

Cited by 16 publications

(9 citation statements)

References 23 publications

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“…Glycerophospholipid catabolism provides lipid polar heads and fatty acids, which could be degraded through β-oxidation, generating energy and biosynthetic precursors via Krebs and glyoxylate cycles and gluconeogenesis (32)(33)(34)(35). Glycerophospholipid turnover has also been verified in related species, such as Mycobacterium smegmatis and Mycobacterium phlei (36). Finally, Rv3842 is essential in vivo during macrophage infection (37).…”

Section: Resultsmentioning

confidence: 99%

Discovery of a glycerol 3-phosphate phosphatase reveals glycerophospholipid polar head recycling in Mycobacterium tuberculosis

Larrouy-Maumus

Biswas

Hunt

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Functional assignment of enzymes encoded by the Mycobacterium tuberculosis genome is largely incomplete despite recent advances in genomics and bioinformatics. Here, we applied an activitybased metabolomic profiling method to assign function to a unique phosphatase, Rv1692. In contrast to its annotation as a nucleotide phosphatase, metabolomic profiling and kinetic characterization indicate that Rv1692 is a D,L-glycerol 3-phosphate phosphatase. Crystal structures of Rv1692 reveal a unique architecture, a fusion of a predicted haloacid dehalogenase fold with a previously unidentified GCN5-related N-acetyltransferase region.Although not directly involved in acetyl transfer, or regulation of enzymatic activity in vitro, this GCN5-related N-acetyltransferase region is critical for the solubility of the phosphatase. Structural and biochemical analysis shows that the active site features are adapted for recognition of small polyol phosphates, and not nucleotide substrates. Functional assignment and metabolomic studies of M. tuberculosis lacking rv1692 demonstrate that Rv1692 is the final enzyme involved in glycerophospholipid recycling/catabolism, a pathway not previously described in M. tuberculosis.haloacid dehalogenase superfamily | enzyme function | pathway discovery E ach year, 1.4 million people succumb to tuberculosis, making Mycobacterium tuberculosis the deadliest bacterium affecting mankind (1). In addition, the dissemination of strains resistant to several antibiotics underscores the need for better understanding of this pathogen and for the development of novel vaccines and therapeutics (2, 3). Our approach to elucidating the unique pervasiveness of M. tuberculosis is through comprehensive discovery and characterization of metabolic pathways, as metabolism underlies survival of the bacteria both inside and outside the host and can contribute to phenotypic and genetic drug resistance.The M. tuberculosis genome encodes for 4,043 genes, of which 3,933 encode proteins (4, 5). Many genes with essential functions, such as DNA replication, protein and RNA synthesis, and cell-division, have close homologs in other bacteria, and their functions are annotated largely on the basis of analysis of their counterparts. However, functions of at least one-third of the genes are unknown or putative (4). In particular, little is known about genes that are not conserved or conditionally important. Characterization of such genes presents a daunting task. M. tuberculosis is thought to be subjected to a myriad of conditions during its life cycle in the host, such as low pH, reactive oxygen and nitrogen species, and so on (6, 7). Understanding how M. tuberculosis adapts to and even thrives in such diverse environments is critical to our understanding of the pathology itself and for the discovery of novel therapeutics to treat tuberculosis.We applied an innovative, activity-based metabolomic profiling (ABMP) approach to assign function to orphan (without a priori known substrates/products) mycobacterial enzymes and to discover unk...

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Section: Resultsmentioning

confidence: 99%

Discovery of a glycerol 3-phosphate phosphatase reveals glycerophospholipid polar head recycling in Mycobacterium tuberculosis

Larrouy-Maumus

Biswas

Hunt

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…The extraction of total lipids from whole cells using chloroform∶methanol (2∶1 v/v) and the separation of polar lipids in a solvent system containing chloroform∶methanol∶water (65∶25∶4) in the first dimension and chloroform∶methanol∶acetic acid∶water (80∶12∶16∶4) in the second dimension were performed as described previously [7],[32],[39]. Polar lipids were visualized by exposing the plates to iodine vapors or staining them with ninhydrin in order to detect amino acid-containing lipids.…”

Section: Methodsmentioning

confidence: 99%

“…Mtb PLs are known to function as important immune modulators [4] and have been shown to be released within phagosomes and transferred into lysosomes [5],[6]. It is interesting to note that PG, which is an abundant PL in other bacteria, is only a minor species in Mycobacteria, whereas CL is a major species [2],[3] with a high turnover rate [7].…”

Section: Introductionmentioning

confidence: 99%

The Two-Domain LysX Protein of Mycobacterium tuberculosis Is Required for Production of Lysinylated Phosphatidylglycerol and Resistance to Cationic Antimicrobial Peptides

et al. 2009

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The well-recognized phospholipids (PLs) of Mycobacterium tuberculosis (Mtb) include several acidic species such as phosphatidylglycerol (PG), cardiolipin, phosphatidylinositol and its mannoside derivatives, in addition to a single basic species, phosphatidylethanolamine. Here we demonstrate that an additional basic PL, lysinylated PG (L-PG), is a component of the PLs of Mtb H37Rv and that the lysX gene encoding the two-domain lysyl-transferase (mprF)-lysyl-tRNA synthetase (lysU) protein is responsible for L-PG production. The Mtb lysX mutant is sensitive to cationic antibiotics and peptides, shows increased association with lysosome-associated membrane protein–positive vesicles, and it exhibits altered membrane potential compared to wild type. A lysX complementing strain expressing the intact lysX gene, but not one expressing mprF alone, restored the production of L-PG and rescued the lysX mutant phenotypes, indicating that the expression of both proteins is required for LysX function. The lysX mutant also showed defective growth in mouse and guinea pig lungs and showed reduced pathology relative to wild type, indicating that LysX activity is required for full virulence. Together, our results suggest that LysX-mediated production of L-PG is necessary for the maintenance of optimal membrane integrity and for survival of the pathogen upon infection.

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“…With the cell wall lipids making up about 10% of the dry weight of mycobacterium (Dhariwal et al, 1976), wall and envelope biosynthesis represents a massive effort on the part of Mycobacterium tuberculosis and other Mycobacteria spp. Occupying a central position in nucleic acid metabolism as well as cell wall biosynthesis is the availability of a large, albeit carefully regulated, pool of NTPs and dNTPs.…”

Section: Introductionmentioning

confidence: 99%

The nucleoside diphosphate kinase of Mycobacterium smegmatis: identification of proteins that modulate specificity of nucleoside triphosphate synthesis by the enzyme

Shankar

Hershberger

Chakrabarty

1997

Molecular Microbiology

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SummaryWe report the purification and characterization of the enzyme nucleoside diphosphate kinase (Ndk) from Mycobacterium smegmatis. The N-terminus of the enzyme was blocked but an internal sequence showed approx. 70% homology with the same enzymes from Pseudomonas aeruginosa and Escherichia coli. Immobilization of the mycobacterial nucleoside diphosphate kinase on a Sepharose 4B matrix and passing the total cell extract through it revealed four proteins (P 70 , P 65 , P 60 , and P 50 , respectively) of M r 70 kDa, 65 kDa, 60 kDa and 50 kDa that were retained by the column. While the proteins of M r 70 kDa and 50 kDa modulated the activity of Ndk directing it towards GTP synthesis, the 60 kDa protein channelled the specificity of Ndk entirely towards CTP synthesis. The 65 kDa protein modulated the specificity of Ndk directing it entirely towards UTP synthesis. The specificity for such mycobacterial proteins towards NTP synthesis is retained when they are complexed with P. aeruginosa Ndk. We further demonstrate that the P 70 protein is pyruvate kinase and that each of the four proteins forms a complex with Ndk and alters its substrate specificity. Given the ubiquitous nature of Ndk in the living cell and its role in maintaining correct ratios of intracellular nucleoside triphosphates, the implications of the occurrence of these complexes have been discussed in relation to the precursor pool for cell wall biosynthesis as well as RNA/DNA synthesis.

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Turnover of lipids in Mycobacterium smegmatis CDC 46 and Mycobacterium phlei ATCC 354

Cited by 16 publications

References 23 publications

Discovery of a glycerol 3-phosphate phosphatase reveals glycerophospholipid polar head recycling in Mycobacterium tuberculosis

Discovery of a glycerol 3-phosphate phosphatase reveals glycerophospholipid polar head recycling in Mycobacterium tuberculosis

The Two-Domain LysX Protein of Mycobacterium tuberculosis Is Required for Production of Lysinylated Phosphatidylglycerol and Resistance to Cationic Antimicrobial Peptides

The nucleoside diphosphate kinase of Mycobacterium smegmatis: identification of proteins that modulate specificity of nucleoside triphosphate synthesis by the enzyme

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