title: Cloning, expression, purification and kinetics of trehalose-6-phosphate phosphatase of filarial parasite Brugia malayi.
abstract: The pleiotropic functions of disaccharide trehalose in the biology of nematodes and its absence from mammalian cells suggest that its biosynthesis may provide a useful target for developing novel nematicidal drugs. The trehalose-6-phosphate phosphatase (TPP), one of the enzymes of trehalose metabolism has not been characterized so far in nematodes except the free living nematode Caenorhabditis elegans where it's silencing results into lethal outcomes. This prompted us to clone and characterize Brugia malayi TPP in order to discover novel antifilarial drug target. The recombinant protein (Bm-TPP) was purified with apparent homogeneity on a metal ion column and it was found to possess high phosphatase activity with robust specificity for the substrate trehalose-6-phosphate. Bm-TPP was found to be a member of the HAD-like hydrolase super family II based on the conserved motifs required for catalytic reaction. The K(m) for substrate trehalose-6-phosphate was around 0.42 mM with pH optimum ∼7.0 and the enzyme showed an almost absolute requirement for Mg(2+) as a metal ion. Bm-TPP was expressed in all the life-stages of B. malayi. In the absence of an effective macrofilaricidal agent and validated antifilarial drug target, Bm-TPP bodes well as a rational drug target against lymphatic filariasis.
authors.affiliation: Division of Parasitology, Central Drug Research Institute, Lucknow, UP, India.
authors.forename: Susheela
authors.initials: S
authors.lastname: Kushwaha
authors.forename: Prashant K
authors.initials: PK
authors.lastname: Singh
authors.forename: Ajay K
authors.initials: AK
authors.lastname: Rana
authors.forename: Shailja
authors.initials: S
authors.lastname: Misra-Bhattacharya
mesh_terms: D000818:Animals; D017178:Brugia malayi / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000378:metabolism; D014199:Trehalose / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D003067:Coenzymes; D011994:Recombinant Proteins; D013403:Sugar Phosphates; C082722:trehalose-6-phosphate; D014199:Trehalose; C023178:trehalose-phosphatase; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium