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...