title: 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 degrees C for up to 6 min, but was slowly inactivated at 70 degrees C. Circular dichroism studies on recombinant TPP indicate that the secondary structure of this protein has considerable beta-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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
authors.forename: Stacey
authors.initials: S
authors.lastname: Klutts
authors.forename: Irena
authors.initials: I
authors.lastname: Pastuszak
authors.forename: Vineetha Koroth
authors.initials: VK
authors.lastname: Edavana
authors.forename: Prajitha
authors.initials: P
authors.lastname: Thampi
authors.forename: Yuan-Tseng
authors.initials: YT
authors.lastname: Pan
authors.forename: Edathera C
authors.initials: EC
authors.lastname: Abraham
authors.forename: J David
authors.initials: JD
authors.lastname: Carroll
authors.forename: Alan D
authors.initials: AD
authors.lastname: Elbein
mesh_terms: D000595:Amino Acid Sequence; D002412:Cations; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000201:enzymology*; D009844:Oligosaccharides / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D002412:Cations; D003902:Detergents; D009844:Oligosaccharides; D010743:Phospholipids; D011994:Recombinant Proteins; C026602:diumycin; C023178:trehalose-phosphatase; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium; C419802:moenomycin