Application | Comment | Organism |
---|---|---|
drug development | enzyme MtbTPP (OtsB2) is considered a promising potential target for discovery of antimicrobial drugs | Mycobacterium tuberculosis |
Cloned (Comment) | Organism |
---|---|
gene Rv3372, sequence comparisons, recombinant expression of wild-type enzyme and selenomethionine-substituted enzyme mutant MtbTPP-M1 in Escherichia coli strain BL21(DE3) | Mycobacterium tuberculosis |
Crystallization (Comment) | Organism |
---|---|
purified apo-active enzyme and trehalose-6-phosphate-complexed enzyme, vapor diffusion technique, mixing of 0.001 ml of 15 mg/ml protein in 20 mM Tris-HCl, pH 7.0, 50 mM NaCl, 1 mM MgCl2, and 10 mM DTT, with 0.001 ml of reservoir solution containing 20 mM sodium acetate trihydrate, pH 4.6, 50 mM CaCl2, 5% 2-propanol, 160 mM ammonium formate, and 16% PEG 3350, microseeding strategy, for substrate-bound crystals, soaking in 50 mM trehalose-6-phosphate for 6 to 12 h, 7 days, 20°C, X-ray diffraction structure detremination and analysis at 2.6 A and 1.7 A resolution, respectively | Mycobacterium tuberculosis |
Protein Variants | Comment | Organism |
---|---|---|
D147A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Mycobacterium tuberculosis |
D149A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Mycobacterium tuberculosis |
D330A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Mycobacterium tuberculosis |
D331A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Mycobacterium tuberculosis |
L29M/L133M | site-directed mutagenesis, mutant MtbTPP-M1 | Mycobacterium tuberculosis |
additional information | deletion of the N-terminal domain results in mutant DELTAN-terminal domaincomprising residues 121-391, which shows 70% reduced activity compared to wild-type, deletion of residues 121-391 resulting in the isolated N-terminal domain and comprising residues 1-120 leads to complete loss of activity | Mycobacterium tuberculosis |
S68A | site-directed mutagenesis, the mutant shows activity similar to the wild-type enzyme | Mycobacterium tuberculosis |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required, Asp147 and Asp149, both located in motif I near the C terminus of beta8, coordinate the Mg2+ via their carboxylate group OD2 and carbonyl oxygen, respectively. Asp330 in motif III is involved in the coordination of the Mg2+ with its side chain. Mg2+ is also coordinated by another catalytically important and conserved residue for the HAD superfamily members, Asp331 in motif III, via a water molecule bridge | Mycobacterium tuberculosis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
alpha,alpha-trehalose 6-phosphate + H2O | Mycobacterium tuberculosis | - |
alpha,alpha-trehalose + phosphate | - |
? | |
alpha,alpha-trehalose 6-phosphate + H2O | Mycobacterium tuberculosis H37Rv | - |
alpha,alpha-trehalose + phosphate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Mycobacterium tuberculosis | P9WFZ5 | - |
- |
Mycobacterium tuberculosis H37Rv | P9WFZ5 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type enzyme and selenomethionine-substituted enzyme mutant MtbTPP-M1 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, dialysis, and anion exchange chromatography, followed by gel filtration | Mycobacterium tuberculosis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
alpha,alpha-trehalose 6-phosphate + H2O | - |
Mycobacterium tuberculosis | alpha,alpha-trehalose + phosphate | - |
? | |
alpha,alpha-trehalose 6-phosphate + H2O | - |
Mycobacterium tuberculosis H37Rv | alpha,alpha-trehalose + phosphate | - |
? | |
additional information | substrate binding structure analysis, overview. The substrate binding site of MtbTPP is located within a highly positively charged cavity formed by the 3 conserved motifs. In the complex structure, the sugar group of trehalose 6-phosphate is likely to localize to the opening of the cleft between the hydrolase domain and the cap domain. In contrast, the phosphate group is buried inside the MtbTPP active site and close to the Mg2+. Four MtbTPP residues directly participate in the stabilization of the substrate: Asp149, Ser185, and Gly186, forming hydrogen bonds with the 3 phosphoryl oxygen atoms of trehalose 6-phosphate, and Val156, interacting with the sugar group via its main chain amide | Mycobacterium tuberculosis | ? | - |
? | |
additional information | substrate binding structure analysis, overview. The substrate binding site of MtbTPP is located within a highly positively charged cavity formed by the 3 conserved motifs. In the complex structure, the sugar group of trehalose 6-phosphate is likely to localize to the opening of the cleft between the hydrolase domain and the cap domain. In contrast, the phosphate group is buried inside the MtbTPP active site and close to the Mg2+. Four MtbTPP residues directly participate in the stabilization of the substrate: Asp149, Ser185, and Gly186, forming hydrogen bonds with the 3 phosphoryl oxygen atoms of trehalose 6-phosphate, and Val156, interacting with the sugar group via its main chain amide | Mycobacterium tuberculosis H37Rv | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
? | x * 43000, recombinant His-tagged enzyme, SDS-PAGE | Mycobacterium tuberculosis |
More | the N-terminal domain (residues 1-120) contains 4 alpha-helices (alpha1-alpha4), 7 beta-stands (beta1-beta7), and one 310 helix (eta1). Hydrolase domain, N-terminal domain, and cap domain structures are in an alphabetaalpha fold, with the central 7-stranded beta-sheet encompassed by helices alpha1 to alpha4 and eta1. The 3 domains are connected by loops of various lengths, overview | Mycobacterium tuberculosis |
Synonyms | Comment | Organism |
---|---|---|
MtbTPP | - |
Mycobacterium tuberculosis |
otsB2 | - |
Mycobacterium tuberculosis |
trehalose 6-phosphate phosphatase | - |
Mycobacterium tuberculosis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Mycobacterium tuberculosis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at | Mycobacterium tuberculosis |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to the haloacid dehalogenase (HAD) superfamily, which includes various phosphatases, epoxide hydrolases, P-type ATPases, and L-2-haloacid dehalogenases. The ubiquitous HAD superfamily features 2 domains: the core domain and the cap domain. Both domains are notably conserved across HAD superfamily, while the cap domain typically varies in size | Mycobacterium tuberculosis |
additional information | residues interacting with the substrate in catalysis are Asp147, Asp149, Asp330, and Asp331, they are pivotal for the enzymatic activity of enzyme MtbTPP. Enzyme structure comparisons, overview | Mycobacterium tuberculosis |
physiological function | trehalose-6-phosphate phosphatase (MtbTPP), an essential enzyme in the trehalose biosynthesis OtsAB pathway, catalyzes the dephosphorylation of trehalose-6-phosphate to generate trehalose, and plays a critical role in Mycobacterium tuberculosis survival-associated cell wall formation and permeability | Mycobacterium tuberculosis |