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glpX Gene of Mycobacterium tuberculosis: Heterologous Expression, Purification, and Enzymatic Characterization of the Encoded Fructose 1,6-bisphosphatase II

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Title: glpX Gene of Mycobacterium tuberculosis: Heterologous Expression, Purification, and Enzymatic Characterization of the Encoded Fructose 1,6-bisphosphatase II
Author(s): Gutka, Hiten J.; Rukseree, Kamolchanok; Wheeler, Paul R.; Franzblau, Scott G.; Movahedzadeh, Farahnaz
Subject(s): glpX Fructose 1 6–bisphosphatase Mycobacterium tuberculosis Gluconeogenesis
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 2 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.
Issue Date: 2011-03-31
Publisher: Humana Press
Citation Info: Gutka, H. J., Rukseree, K., Wheeler, P. R., Franzblau, S. G., & Movahedzadeh, F. 2011. glpX Gene of Mycobacterium tuberculosis: Heterologous Expression, Purification, and Enzymatic Characterization of the Encoded Fructose 1,6-bisphosphatase II. Applied Biochemistry and Biotechnology. DOI: 10.1007/s12010-011-9219-x
Type: Article
Description: Post print version of article may differ from published version. The original publication is available at springerlink.com; DOI: 10.1007/s12010-011-9219-x.
URI: http://hdl.handle.net/10027/7706
ISSN: 0273-2289
Sponsor: The authors are thankful to American Lung Association (Grant No. RG-82534-N).
Date Available in INDIGO: 2011-05-27
 

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