Enzymatic Characterization of Fructose 1,6-Bisphosphatase II from Francisella tularensis, an Essential Enzyme for Pathogenesis

• Published in: Applied biochemistry and biotechnology 2017-12, Vol.183 (4), p.1439-1454
• Main Authors: Gutka, Hiten J., Wolf, Nina M., Bondoc, Jasper Marc G., Movahedzadeh, Farahnaz
• Format: Article
• Language: English
• Subjects: Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biochemistry; Biotechnology; Chemistry; Chemistry and Materials Science; Complementation; Deactivation; Drug development; E coli; Enzymatic activity; Enzymes; Escherichia coli - enzymology; Escherichia coli - genetics; Francisella tularensis; Francisella tularensis - enzymology; Francisella tularensis - genetics; Francisella tularensis - pathogenicity; Fructose; Fructose-Bisphosphatase - chemistry; Fructose-Bisphosphatase - genetics; Fructose-Bisphosphatase - metabolism; Genetic Complementation Test; Glycerol; Inactivation; Pathogenesis; Phosphates; Purification; Substrates
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-017-2512-6

The glpX gene from Francisella tularensis encodes for the class II fructose 1,6-bisphosphatase (FBPaseII) enzyme. The glpX gene has been verified to be essential in F. tularensis , and the inactivation of this gene leads to impaired bacterial growth on gluconeogenic substrates. In the present work, we have complemented a ∆glpX mutant of Escherichia coli with the glpX gene of F. tularensis (FTF1631c). Our complementation work independently verifies that the glpX gene (FTF1631c) in F. tularensis is indeed an FBPase and supports the growth of the ΔglpX E. coli mutant on glycerol-containing media. We have performed heterologous expression and purification of the glpX encoded FBPaseII in F. tularensis . We have confirmed the function of glpX as an FBPase and optimized the conditions for enzymatic activity. Mn 2+ was found to be an absolute requirement for activity, with no other metal substitutions rendering the enzyme active. The kinetic parameters for this enzyme were found as follows: K m 11 μM, V max 2.0 units/mg, k cat 1.2 s −1 , k cat /K m 120 mM −1  s −1 , and a specific activity of 2.0 units/mg. Size exclusion data suggested an abundance of a tetrameric species in solution. Our findings on the enzyme’s properties will facilitate the initial stages of a structure-based drug design program targeting this essential gene of F. tularensis .