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, gene products of the glp regulon mediate utilization of glycerol and sn-glycerol 3-phosphate. The glpFKX operon encodes glycerol diffusion facilitator, glycerol kinase, and as shown here, a fructose 1,6-bisphosphatase that is distinct from the previously described fbp-encoded enzyme. The purified enzyme was dimeric, dependent on Mn 2؉ for activity, and exhibited an apparent K m of 35 M for fructose 1,6-bisphosphate. The enzyme was inhibited by ADP and phosphate and activated by phosphoenolpyruvate.Growth of Escherichia coli on glycerol or sn-glycerol 3-phosphate (glycerol-P) as the sole carbon source is mediated primarily by the glp regulon (15). The glpFKX operon, one of the five operons of the regulon, encodes glycerol facilitator (glpF), glycerol kinase (glpK), and a protein of unknown function (glpX) (31, 32). It was initially reported that GlpX displays limited sequence similarity to the Synechococcus leopoliensis fructose 1,6-bisphosphatase (FBPase) (31). In our work, a more recent BLAST search revealed that GlpX manifests 39% identity to an FBPase of Synechococcus sp. strain PCC7492 (29). Until now, the only recognized E. coli FBPase was encoded by fbp (25). This FBPase (FBPase I) has only 10% identity to the amino acid sequence of glpX-encoded FBPase (FBPase II). E. coli FBPase I is dependent on Mg 2ϩ , is inhibited by low levels of AMP, is tetrameric (1), and is necessary for growth of E. coli on gluconeogenic substrates such as glycerol or succinate (10).It is not clear why E. coli would maintain two distinct FBPases. FBPases can modulate the concentration of fructose 1,6-bisphosphate [Fru(1,6)P 2 ] and fructose 6-phosphate. These two regulatory hexoses affect glycolysis enzymes 6-phosphofructokinases I and II, pyruvate kinase I, and phosphoenolpyruvate (PEP) carboxylase (2,4,13,20); glycogen synthesis enzyme ADP-glucose pyrophosphorylase (12); and carbonsource import pathway enzymes glycerol kinase and 1-phosphofructokinase (6, 15). Flux through the Embden-Meyerhof pathway in the direction of glycolysis or gluconeogenesis can be allosterically controlled at the enzyme level by other metabolites as well: PEP, ATP, ADP or AMP (9). The potential "futile cycle" of phosphofructokinases and FBPases is also alleviated by this regulation. Therefore, regulation of FBPases is important.In this communication, the FBPase activity of the glpX gene product is documented. The glpX-encoded enzyme, FBPase II, was purified and characterized, enabling comparison of the attributes of E. coli FBPases in vitro. Further, a chromosomal insertion mutation in glpX was constructed to test the physiological effects of the glpX mutation on carbohydrate metabolism.E. coli strains and cloning of glpX. E. coli strains used in this study are listed in Table 1. Strains were grown in Luria broth (LB) supplemented with antibiotics as needed or in minimal medium (7) containing 0.4% glycerol or 0.2% glucose.The glpX gene was PCR amplified from chromosomal DNA of strain MG1655 using the primer pair acgtgaaTTCCCCTG TGCT...

Structural and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli

“…Kinetic parameters were determined by nonlinear curve fitting from the Lineweaver-Burk plot using GraphPad Prism software (version 4.00 for Windows, GraphPad Software, San Diego, CA). The effect of inorganic phosphate on FBPase activity of GlpX and YggF was measured using the continuous assay with phosphoglucoisomerase and glucose-6-phosphate dehydrogenase (9).…”

“…The E. coli GlpX, a class II enzyme FBPase, has been shown to possess a Mn 2ϩ -dependent FBPase activity (9). The increased expression of glpX from a multicopy plasmid complemented the Fbp Ϫ phenotype; however, the glpX knock-out strain grew normally on gluconeogenic substrates (succinate or glycerol) (9).…”

“…This enzyme has been characterized both biochemically and structurally and shown to be inhibited by low concentrations of AMP (IC 50 15 M) (11,29,30). The E. coli GlpX, a class II enzyme FBPase, has been shown to possess a Mn 2ϩ -dependent FBPase activity (9). The increased expression of glpX from a multicopy plasmid complemented the Fbp Ϫ phenotype; however, the glpX knock-out strain grew normally on gluconeogenic substrates (succinate or glycerol) (9).…”

“…Types I, II, and III are primarily in bacteria, type IV in archaea (a bifunctional FBPase/ inositol monophosphatase), and type V in thermophilic prokaryotes from both domains (11). Many organisms have more than one FBPase, mostly the combination of types I ϩ II or II ϩ III, but no bacterial genome has a combination of types I and III FBPases (9). The type I FBPase is the most widely distributed among living organisms and is the primary FBPase in Esche-richia coli, most bacteria, a few archaea, and all eukaryotes (9,(11)(12)(13)(14)(15).…”

“…The type I FBPase is the most widely distributed among living organisms and is the primary FBPase in Esche-richia coli, most bacteria, a few archaea, and all eukaryotes (9,(11)(12)(13)(14)(15). The type II FBPases are represented by the E. coli GlpX and FBPase F-I from Synechocystis PCC6803 (9,16); type III is represented by the Bacillus subtilis FBPase (17); type IV is represented by the dual activity FBPases/inosine monophosphatases FbpA from Pyrococcus furiosus (18), MJ0109 from Methanococcus jannaschii (19), and AF2372 from Archaeoglobus fulgidus (20); and type V is represented by the FBPases TK2164 from Pyrococcus (Thermococcus) kodakaraensis and ST0318 from Sulfolobus tokodai (10,21).…”