Cloning and expression of the trehalose-phosphate phosphatase of Mycobacterium tuberculosis: comparison to the enzyme from Mycobacterium smegmatis

Edavana, Vineetha Koroth; Pastuszak, Irena; Carroll, J.; Thampi, Prajitha; Abraham, Edathera C; Elbein, Alan D.

doi:10.1016/j.abb.2004.02.014

Cited by 28 publications

(24 citation statements)

References 31 publications

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“…This result confirms that failure to isolate the otsB2 mutant is because of the fact that this gene has an essential role for in vitro replication of M. tuberculosis. This role cannot be performed by the otsB1 gene, and the lack of phenotype for the ⌬otsB1 clone is consistent with the biochemical observation that the protein product of otsB1 lacks functional trehalose-6-phosphate phosphatase activity (17).…”

Section: Construction Of M Tuberculosis Mutants With Deletions Insupporting

confidence: 84%

“…In contrast to the results with M. smegmatis and with C. glutamicum, our studies demonstrated that OtsAB is the predominant pathway in M. tuberculosis. The mutagenesis results are consistent with biochemical studies demonstrating that trehalose-6-phosphate phosphatase activity is associated with only one of the OtsB homologues in M. tuberculosis (17) and highlight this enzyme as an attractive target for drug development.…”

supporting

confidence: 86%

“…The lethal phenotype of the ⌬otsB2 mutant demonstrates that the phosphatase function of OtsB cannot be arrived at by other enzymes, further reinforcing the conclusion from biochemical studies that open reading frame Rv2006, annotated as otsB1 in the genome, is inactive in this respect (17). Trehalose-6-phosphate acts as an important signal for metabolic control in Saccharomyces cerevisiae, where it regulates the first steps in glycolysis through the inhibition of hexokinase II, and its accumulation is detrimental to the organism (29).…”

Section: Fig 2 Mortality Counts For Mice Infected With 10supporting

confidence: 68%

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The OtsAB Pathway Is Essential for Trehalose Biosynthesis in Mycobacterium tuberculosis

Murphy

Stewart

Mischenko

et al. 2005

Journal of Biological Chemistry

130

137

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The disaccharide trehalose is the major free sugar in the cytoplasm of mycobacteria; it is a constituent of cell wall glycolipids, and it plays a role in mycolic acid transport during cell wall biogenesis. The pleiotropic role of trehalose in the biology of Mycobacterium tuberculosis and its absence from mammalian cells suggests that its biosynthesis may provide a useful target for novel drugs. However, there are three potential pathways for trehalose biosynthesis in M. tuberculosis, and the aim of the present study was to introduce mutations into each of the pathways to determine whether or not they are functionally redundant. The results show that the OtsAB pathway, which generates trehalose from glucose and glucose-6-phosphate, is the dominant pathway required for M. tuberculosis growth in laboratory culture and for virulence in a mouse model. Of the two otsB homologues annotated in the genome sequence of M. tuberculosis, only OtsB2 (Rv3372) has a functional role in the pathway. OtsB2, trehalose-6-phosphate phosphatase, is strictly essential for growth and provides a tractable target for high throughput screening. Inactivation of the TreYZ pathway, which can generate trehalose from ␣-1,4-linked glucose polymers, had no effect on the growth of M. tuberculosis in vitro or in mice. Deletion of the treS gene altered the late stages of pathogenesis of M. tuberculosis in mice, significantly increasing the time to death in a chronic infection model. Because the TreS enzyme catalyzes the interconversion of trehalose and maltose, the mouse phenotype could reflect either a requirement for synthesis of additional trehalose or, conversely, a requirement for breakdown of stored trehalose to liberate free glucose.

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Section: Construction Of M Tuberculosis Mutants With Deletions Insupporting

confidence: 84%

supporting

confidence: 86%

Section: Fig 2 Mortality Counts For Mice Infected With 10supporting

confidence: 68%

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The OtsAB Pathway Is Essential for Trehalose Biosynthesis in Mycobacterium tuberculosis

Murphy

Stewart

Mischenko

et al. 2005

Journal of Biological Chemistry

130

137

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“…The important biological functions of trehalose combined with the fact that humans do not biosynthesize this sugar initially raised interest in its biosynthetic enzymes as targets for the development of novel antituberculosis drugs (12). However, the realization that there are three biosynthetic pathways for trehalose in M. tuberculosis has tempered enthusiasm for this approach (13).…”

mentioning

confidence: 99%

“…coli.) (12). The second pathway (TreY-TreZ) utilizes glycogen or maltooligosaccharides as the starting material.…”

mentioning

confidence: 99%

Mechanistic Analysis of Trehalose Synthase from Mycobacterium smegmatis

Zhang

Pan

et al. 2011

Journal of Biological Chemistry

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Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose and trehalose and has been shown recently to function primarily in the mobilization of trehalose as a glycogen precursor. Consequently, the mechanism of this intriguing isomerase is of both academic and potential pharmacological interest. TreS catalyzes the hydrolytic cleavage of ␣-aryl glucosides as well as ␣-glucosyl fluoride, thereby allowing facile, continuous assays. Reaction of TreS with 5-fluoroglycosyl fluorides results in the trapping of a covalent glycosyl-enzyme intermediate consistent with TreS being a member of the retaining glycoside hydrolase family 13 enzyme family, thus likely following a two-step, double displacement mechanism. This trapped intermediate was subjected to protease digestion followed by LC-MS/MS analysis, and Asp 230 was thereby identified as the catalytic nucleophile. The isomerization reaction was shown to be an intramolecular process by demonstration of the inability of TreS to incorporate isotope-labeled exogenous glucose into maltose or trehalose consistent with previous studies on other TreS enzymes. The absence of a secondary deuterium kinetic isotope effect and the general independence of k cat upon leaving group ability both point to a rate-determining conformational change, likely the opening and closing of the enzyme active site.

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Inhibition of Trehalose Synthesis in Lepidoptera Reduces Larval Fitness

Tellis,

Mohite,

Nair

et al. 2023

Advanced Biology

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Trehalose is synthesized in insects through the trehalose 6‐phosphate synthase and phosphatase (TPS/TPP) pathway. TPP dephosphorylates trehalose 6‐phosphate to release trehalose. Trehalose is involved in metamorphosis, but its relation with body weight, size, and developmental timing is unexplored. The expression and activity of TPS/TPP fluctuate depending on trehalose demand. Thus, TPS/TPP inhibition can highlight the significance of trehalose in insect physiology. TPS/TPP transcript levels are elevated in the pre‐pupal and pupal stages in Helicoverpa armigera. The inhibition of recombinantly expressed TPP by N‐(phenylthio)phthalimide (NPP), is validated by in vitro assays. In vivo inhibition of trehalose synthesis reduces larval weight and size, hampers metamorphosis, and reduces its overall fitness. Insufficient trehalose leads to a shift in glucose flux, reduced energy, and dysregulated fatty acid oxidation. Metabolomics reaffirms the depletion of trehalose, glucose, glucose 6‐phosphate, and suppressed tricarboxylic acid cycle. Reduced trehalose hampers the energy level affecting larval vitality. Through trehalose synthesis inhibition, the importance of trehalose in insect physiology and development is investigated. Also, in two other lepidopterans, TPP inhibition impedes physiology and survival. NPP is also found to be effective as an insecticidal formulation. Overall, trehalose levels affect the larval size, weight, and metabolic homeostasis for larval‐pupal transition in lepidoptera.

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Cloning and expression of the trehalose-phosphate phosphatase of Mycobacterium tuberculosis: comparison to the enzyme from Mycobacterium smegmatis

Cited by 28 publications

References 31 publications

The OtsAB Pathway Is Essential for Trehalose Biosynthesis in Mycobacterium tuberculosis

The OtsAB Pathway Is Essential for Trehalose Biosynthesis in Mycobacterium tuberculosis

Mechanistic Analysis of Trehalose Synthase from Mycobacterium smegmatis

Inhibition of Trehalose Synthesis in Lepidoptera Reduces Larval Fitness

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