Purification, Cloning, Expression, and Properties of Mycobacterial Trehalose-phosphate Phosphatase

Abstract

The trehalose-phosphate phosphatase (TPP) was purified from the cytosol of Mycobacterium smegmatis to near homogeneity using a variety of conventional steps to achieve a purification of about 1600-fold with a yield of active enzyme of about 1%. Based on gel filtration, the active enzyme had a molecular weight of about 27,000, and the most purified fraction also gave a major band on SDS-PAGE corresponding to a molecular weight of about 27,000. A number of peptides from the 27-kDa protein were sequenced and these sequences showed considerable homology to the trehalose-P phosphatase (otsB) of Escherichia coli. Based on these peptides, the M. smegmatis gene for TPP was cloned and expressed in E. coli. The recombinant protein was synthesized with a (His) 6 tag at the amino terminus. Most of the TPP activity in the crude E. coli sonicate was initially found in the membrane fraction, but it became solubilized in the presence of 0.2% Sarkosyl. The solubilized protein was purified to apparent homogeneity on a metal ion column and this fraction had high phosphatase activity that was completely specific for trehalose-P. The purified enzyme, either isolated from M. smegmatis, or expressed in E. coli, rapidly dephosphorylated trehalose-6-P, but had essentially no activity on any other sugar phosphates, or on p-nitrophenyl phosphate. The K m for trehalose-6-P was about 1.6 mM, and the pH optimum was about 7.5. The native enzyme showed an almost absolute requirement for Mg 2؉ and was not very active with Mn 2؉ , whereas both of these cations were equally effective with the recombinant TPP. The enzyme activity was inhibited by the antibiotics, diumycin and moenomycin, but not by a number of other antibiotics or trehalose analogs. TPP activity was strongly inhibited by the detergents, Sarkosyl and deoxycholate, even at 0.025%, but it was not inhibited by Nonidet P-40, Triton X-100, or octyl glucoside, even at concentrations up to 0.3%. The purified enzyme was stable to heating at 60°C for up to 6 min, but was slowly inactivated at 70°C. Circular dichroism studies on recombinant TPP indicate that the secondary structure of this protein has considerable ␤-pleated sheet and is very compact. TPP may play a key role in the biosynthesis of trehalose compounds, such as trehalose mycolates, and therefore may represent an excellent target site for chemotherapy against tuberculosis and other mycobacterial diseases.

Last Step in the Conversion of Trehalose to Glycogen

“…This is a colorimetric method for hexose determination, which is done in strong H 2 SO 4 that hydrolyzes maltose-1-P to glucose and then determines the amount of glucose. A second series of aliquots was removed for the measurement of inorganic phosphate using the arsenomolybdate colorimetric method (24). This experiment was repeated several times with very similar results, and 50 to 65% of the maltose-1-P added to the incubations was converted to glycogen as indicated by the loss of maltose-1-P as well as by the amount of radioactivity incorporated into glycogen (see data in Table 4).…”

Kinetic characterization and molecular modeling of trehalose-6-phosphate phosphatase from Anopheles gambiae and expressed in Pichia pastoris

“…The TPP gene was selected because it codes for a protein that is involved in the formation of disaccharide trehalose in the insect's carbohydrate metabolic pathway. TPP was chosen for cloning and expression because its catalytic product is trehalose, which is not essential for mammal cells, and not glucose, which is the major carbohydrate required for numerous mammalian metabolic pathways (Karthik et al, 2011;Klutts et al, 2003;Kormish and McGhee, 2005;Kushwaha et al, 2011).…”

“…Enzyme activity was determined according to a modification of the method by Klutts et al (2003), which used 1 to 8 mM T6P (SigmaAldrich, St. Louis, MO, USA), 2 mM MgCl2, 50 mM Tris-HCl buffer and 1 µl of recombinant enzyme. The reaction was performed for 10 min, and the Michaelis-Menten curve and Lineweaver-Burk plot were obtained.…”

“…The assay for TPP activity was measured by determining the release of inorganic phosphate from T6P according methodology descripted by Edavana et al (2004) and Klutts et al (2003).…”

“…TPP (EC 3.1.3.12) is a member of the superfamily haloacid dehalogenase (HAD) (Collet et al, 1998) and exhibits significant sequence homology with several phosphatases, P-type ATPases (Koonin and Tatusov, 1994) and other members of the family (Rao et al, 2006). TPP displays specific activity for substrate T6P, resulting in trehalose formation in the presence of water, which is characteristic of enzymes of the hydrolase family with phosphatase activity (Elbein, 2009;Klutts et al, 2003;Kormish and McGhee, 2005;Rao et al, 2006).…”