Literature summary for 3.1.3.12 extracted from

Shan, S.; Min, H.; Liu, T.; Jiang, D.; Rao, Z.
Structural insight into dephosphorylation by trehalose 6-phosphate phosphatase (OtsB2) from Mycobacterium tuberculosis (2016), FASEB J., 30, 3989-3996 .

Application

Application	Comment	Organism
drug development	enzyme MtbTPP (OtsB2) is considered a promising potential target for discovery of antimicrobial drugs	Mycobacterium tuberculosis

Cloned(Commentary)

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 (Commentary)

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

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

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

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/ Products (Substrates)

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

Organism	UniProt	Comment	Textmining
Mycobacterium tuberculosis	P9WFZ5	-	-
Mycobacterium tuberculosis H37Rv	P9WFZ5	-	-

Purification (Commentary)

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 and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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

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

Synonyms	Comment	Organism
MtbTPP	-	Mycobacterium tuberculosis
otsB2	-	Mycobacterium tuberculosis
trehalose 6-phosphate phosphatase	-	Mycobacterium tuberculosis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Mycobacterium tuberculosis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	assay at	Mycobacterium tuberculosis

General Information

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