Type of Document	Master's Thesis
Author	Donahue, Janet Lee
Author's Email Address	jadonah2@vt.edu
URN	etd-04272000-16290048
Title	Purification and Characterization of glpX-Encoded Fructose 1,6-Bisphosphatase, a New Enzyme of the Glycerol 3-Phosphate Regulon of Escherichia coli
Degree	Master of Science
Department	Biochemistry and Anaerobic Microbiology
Advisory Committee	Advisor Name Title Dr. Timothy J. Larson Committee Chair Dr. Glenda Gillaspy Committee Member Dr. John L. Hess Committee Member
Keywords	fructose 6-phosphate carbon metabolism 6-bisphosphatase fructose 1 glycerol 3-phosphate regulon
Date of Defense	2000-04-10
Availability	unrestricted
Abstract xmlns:w="urn:schemas-microsoft-com:office:word" xmlns="http://www.w3.org/TR/REC-html40"> In Escherichia coli, the utilization of glycerol and sn-glycerol 3-phosphate is mediated by gene products of the glp regulon.  The regulon encompasses five operons, including the glpFKX operon.  Although glpF and glpK encode glycerol diffusion facilitator and glycerol kinase, respectively, the function of glpX was unknown. style="mso-spacerun: yes">  In the present work, we show that style='mso-bidi-font-style:normal'>glpX encodes a fructose 1,6-bisphosphatase (FBPase), which catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and phosphate. style="mso-spacerun: yes">  The purified FBPase was dimeric, dependent on Mn2+ for activity and exhibited an apparent Km of 35 mM for fructose 1,6-bisphosphate.  The enzyme was inhibited by ADP, ATP and phosphate and activated by PEP. The attributes of the glpX-encoded FBPase were different from those of the previously characterized E. coli FBPase encoded by fbp. style="mso-spacerun: yes">  Mutants deleted in fbp (Dfbp) display a growth-negative phenotype on gluconeogenic carbon sources such as glycerol, indicating the inability of chromosomal style='mso-bidi-font-style:normal'>glpX+ to complement style='mso-bidi-font-style:normal'>Dfbp. style="mso-spacerun: yes">  However, a D style='mso-bidi-font-style:normal'>fbp mutation was complemented by overexpression of glpX+. style="mso-spacerun: yes">  In contrast, a glpX mutant exhibited a growth-positive phenotype on glycerol, glucose or fructose media.  Surprisingly, a double mutant strain glpX pfkA (6-phosphofructokinase I) was more inhibited in growth on glucose and glycerol media than the pfkA parent.  Carbohydrate metabolism in the style='mso-bidi-font-style:normal'>pfkA background may be affected by the glpX-mediated change in fructose 6-phosphate/fructose 1,6-bisphosphate levels. style="mso-spacerun: yes">  FBPase activities of soluble proteins separated by non-denaturing PAGE were visualized, showing a novel (third) FBPase, perhaps encoded by the glpX homolog, yggF.
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