title: Biochemical characterization and ligand-binding properties of trehalose-6-phosphate phosphatase from Mycobacterium tuberculosis.
abstract: Trehalose-6-phosphate phosphatase (TPP) is an essential enzyme for growth of mycobacteria, which has been identified to be a potential anti-tuberculosis drug target. However, the biochemical and ligand-binding properties and the 3D structure of TPP remain unclear so far. In the present study, we expressed the recombinant TPP protein from Mycobacterium tuberculosis (otsB2/Rv3372). Results from the far-ultraviolet circular dichroism experiments indicated that the secondary structure of TPP was rich in α-helix with a lower structural stability (Cm = 2.099 ± 0.134 M). Ligand-binding assay by isothermal titration calorimetry demonstrated that the recombinant TPP protein could bind with trehalose-6-P in the presence of Mg(2+) (Kd = 39.52 ± 1.78 μM) with a molar ratio of 1 : 1. In addition, the 3D structure of TPP was modeled by I-TASSER, indicating that the TPP protein was composed of a hydrolase domain, a cap domain, and an N-terminal domain. Flexible docking was further conducted by using the Simulations/Dock module of the Molecular Operating Environment software. The binding pocket of TPP for both trehalose-6-P and Mg(2+) was determined, which was located on the interface between the hydrolase domain and the cap domain. Asp149, Gly186, Arg187, Arg291, and Glu295 were identified to be the key residues for TPP binding with trehalose-6-P. This work may lay the basis for further structural and functional studies of TPP and TPP-related novel drug development.
authors.affiliation: Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China.
authors.forename: Lina
authors.initials: L
authors.lastname: Shi
authors.forename: Haiping
authors.initials: H
authors.lastname: Zhang
authors.forename: Yu
authors.initials: Y
authors.lastname: Qiu
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.forename: Xueji
authors.initials: X
authors.lastname: Wu
authors.forename: Honghai
authors.initials: H
authors.lastname: Wang
authors.forename: Xuelian
authors.initials: X
authors.lastname: Zhang
authors.forename: Donghai
authors.initials: D
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D008024:Ligands; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013403:Sugar Phosphates / Q000378:metabolism; D014199:Trehalose / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D008024:Ligands; D011994:Recombinant Proteins; D013403:Sugar Phosphates; C082722:trehalose-6-phosphate; D014199:Trehalose; C023178:trehalose-phosphatase; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium
keywords: circular dichroism; isothermal titration calorimetry; structure model; trehalose-6-P; trehalose-6-phosphate phosphatase