title: glpX gene of Mycobacterium tuberculosis: heterologous expression, purification, and enzymatic characterization of the encoded fructose 1,6-bisphosphatase II.
abstract: The glpX gene (Rv1099c) of Mycobacterium tuberculosis (Mtb) encodes Fructose 1,6-bisphosphatase II (FBPase II; EC 3.1.3.11); a key gluconeogenic enzyme. Mtb possesses glpX homologue as the major known FBPase. This study explored the expression, purification and enzymatic characterization of functionally active FBPase II from Mtb. The glpX gene was cloned, expressed and purified using a two step purification strategy including affinity and size exclusion chromatography. The specific activity of Mtb FBPase II is 1.3 U/mg. The enzyme is oligomeric, followed Michaelis-Menten kinetics with an apparent km = 44 μM. Enzyme activity is dependent on bivalent metal ions and is inhibited by lithium and inorganic phosphate. The pH optimum and thermostability of the enzyme have been determined. The robust expression, purification and assay protocols ensure sufficient production of this protein for structural biology and screening of inhibitors against this enzyme.
authors: Institute for Tuberculosis Research (M/C 964), College of Pharmacy, Room 412, University of Illinois at Chicago, 833 S. Wood St, Chicago, IL 60612-7231, USA.
authors: Hiten J
authors: HJ
authors: Gutka
authors: Kamolchanok
authors: K
authors: Rukseree
authors: Paul R
authors: PR
authors: Wheeler
authors: Scott G
authors: SG
authors: Franzblau
authors: Farahnaz
authors: F
authors: Movahedzadeh
mesh_terms: D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002412:Cations / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D008094:Lithium; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism / Q000494:pharmacology; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D002412:Cations; D004791:Enzyme Inhibitors; D008670:Metals; D011994:Recombinant Proteins; D008345:Manganese; D008094:Lithium; D006597:Fructose-Bisphosphatase; D008274:Magnesium; D002118:Calcium