{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/31955"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/31955","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"Purification and Characterization of glpX-Encoded Fructose 1,6-Bisphosphatase, a New Enzyme of the Glycerol 3-Phosphate Regulon of Escherichia coli","abstract":"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. In the present work, we show that glpX encodes a fructose 1,6-bisphosphatase (FBPase), which catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and phosphate. The purified FBPase was dimeric, dependent on Mn2+ for activity and exhibited an apparent Km of 35 μM 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. Mutants deleted in fbp (Δfbp) display a growthnegative phenotype on gluconeogenic carbon sources such as glycerol, indicating the inability of chromosomal glpX+ to complement Δfbp. However, a Δfbp mutation was complemented by overexpression of glpX+. 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 pfkA background may be affected by the glpXmediated change in fructose 6-phosphate/fructose 1,6-bisphosphate levels. FBPase activities of soluble proteins separated by non-denaturing PAGE were visualized, showing a novel (third) FBPase, perhaps encoded by the glpX homolog, yggF.","abstract_html":"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. In the present work, we show that glpX encodes a fructose 1,6-bisphosphatase (FBPase), which catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and phosphate. The purified FBPase was dimeric, dependent on Mn2+ for activity and exhibited an apparent Km of 35 μM 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. Mutants deleted in fbp (Δfbp) display a growthnegative phenotype on gluconeogenic carbon sources such as glycerol, indicating the inability of chromosomal glpX+ to complement Δfbp. However, a Δfbp mutation was complemented by overexpression of glpX+. 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 pfkA background may be affected by the glpXmediated change in fructose 6-phosphate/fructose 1,6-bisphosphate levels. FBPase activities of soluble proteins separated by non-denaturing PAGE were visualized, showing a novel (third) FBPase, perhaps encoded by the glpX homolog, yggF.","abstract_has_math":false,"creators":["Donahue, Janet Lee"],"institution":"Virginia Tech","degree_name":"Master of Science","degree_level":"masters","degree_discipline":"Biochemistry and Anaerobic Microbiology","degree_department":"Biochemistry and Anaerobic Microbiology","school":null,"contributors":[],"advisors":[],"committee_chairs":["Larson, Timothy J."],"committee_members":["Hess, John L.","Gillaspy, Glenda E."],"year":2000,"date_issued":"2000-04-10","date_published":"2000-04-10","updated_at":"2026-07-22T22:19:42Z","subjects":["fructose 6-phosphate","carbon metabolism","6-bisphosphatase","fructose 1","glycerol 3-phosphate regulon"],"languages":[],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-04272000-16290048"],"render_values":[{"text":"etd-04272000-16290048","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/10919/31955","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeechair","label":"Committee Chair","values":["Larson, Timothy J."]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Hess, John L.","Gillaspy, Glenda E."]},{"key":"dc:contributor.department","label":"Department","values":["Biochemistry and Anaerobic Microbiology"]},{"key":"dc:creator","label":"Author","values":["Donahue, Janet Lee"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2014-03-14T20:34:23Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2014-03-14T20:34:23Z","2001-05-01"]},{"key":"dc:date.issued","label":"Date","values":["2000-04-10"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Tech"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biochemistry and Anaerobic Microbiology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["masters"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Virginia Polytechnic Institute and State University"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["fructose 6-phosphate","carbon metabolism","6-bisphosphatase","fructose 1","glycerol 3-phosphate regulon"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["In Copyright"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://rightsstatements.org/vocab/InC/1.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-04272000-16290048"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/10919/31955"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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. In the present work, we show that glpX encodes a fructose 1,6-bisphosphatase (FBPase), which catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and phosphate. The purified FBPase was dimeric, dependent on Mn2+ for activity and exhibited an apparent Km of 35 μM 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. Mutants deleted in fbp (Δfbp) display a growthnegative phenotype on gluconeogenic carbon sources such as glycerol, indicating the inability of chromosomal glpX+ to complement Δfbp. However, a Δfbp mutation was complemented by overexpression of glpX+. 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 pfkA background may be affected by the glpXmediated change in fructose 6-phosphate/fructose 1,6-bisphosphate levels. FBPase activities of soluble proteins separated by non-denaturing PAGE were visualized, showing a novel (third) FBPase, perhaps encoded by the glpX homolog, yggF."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Master of Science"]},{"key":"dc:title","label":"Title","values":["Purification and Characterization of glpX-Encoded Fructose 1,6-Bisphosphatase, a New Enzyme of the Glycerol 3-Phosphate Regulon of Escherichia coli"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Larson, Timothy J."],"dc:contributor.committeemember":["Hess, John L.","Gillaspy, Glenda E."],"dc:contributor.department":["Biochemistry and Anaerobic Microbiology"],"dc:creator":["Donahue, Janet Lee"],"dc:date.accessioned":["2014-03-14T20:34:23Z"],"dc:date.available":["2014-03-14T20:34:23Z","2001-05-01"],"dc:date.issued":["2000-04-10"],"dc:description.abstract":["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. In the present work, we show that glpX encodes a fructose 1,6-bisphosphatase (FBPase), which catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and phosphate. The purified FBPase was dimeric, dependent on Mn2+ for activity and exhibited an apparent Km of 35 μM 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. Mutants deleted in fbp (Δfbp) display a growthnegative phenotype on gluconeogenic carbon sources such as glycerol, indicating the inability of chromosomal glpX+ to complement Δfbp. However, a Δfbp mutation was complemented by overexpression of glpX+. 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 pfkA background may be affected by the glpXmediated change in fructose 6-phosphate/fructose 1,6-bisphosphate levels. FBPase activities of soluble proteins separated by non-denaturing PAGE were visualized, showing a novel (third) FBPase, perhaps encoded by the glpX homolog, yggF."],"dc:description.degree":["Master of Science"],"dc:identifier.other":["etd-04272000-16290048"],"dc:identifier.uri":["http://hdl.handle.net/10919/31955"],"dc:publisher":["Virginia Tech"],"dc:rights":["In Copyright"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:subject":["fructose 6-phosphate","carbon metabolism","6-bisphosphatase","fructose 1","glycerol 3-phosphate regulon"],"dc:title":["Purification and Characterization of glpX-Encoded Fructose 1,6-Bisphosphatase, a New Enzyme of the Glycerol 3-Phosphate Regulon of Escherichia coli"],"dc:type":["Thesis"],"thesis:degree_discipline":["Biochemistry and Anaerobic Microbiology"],"thesis:degree_level":["masters"],"thesis:degree_name":["Master of Science"],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-22T22:19:42Z"}