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

Abstract

Gluconeogenesis is an important metabolic pathway, which produces glucose from noncarbohydrate precursors such as organic acids, fatty acids, amino acids, or glycerol. Fructose-1,6-bisphosphatase, a key enzyme of gluconeogenesis, is found in all organisms, and five different classes of these enzymes have been identified. Here we demonstrate that Escherichia coli has two class II fructose-1,6-bisphosphatases, GlpX and YggF, which show different catalytic properties. We present the first crystal structure of a class II fructose-1,6-bisphosphatase (GlpX) determined in a free state and in the complex with a substrate (fructose 1,6-bisphosphate) or inhibitor (phosphate). The crystal structure of the ligand-free GlpX revealed a compact, globular shape with two ␣/␤-sandwich domains. The core fold of GlpX is structurally similar to that of Li ؉ -sensitive phosphatases implying that they have a common evolutionary origin and catalytic mechanism. The structure of the GlpX complex with fructose 1,6-bisphosphate revealed that the active site is located between two domains and accommodates several conserved residues coordinating two metal ions and the substrate. The third metal ion is bound to phosphate 6 of the substrate. Inorganic phosphate strongly inhibited activity of both GlpX and YggF, and the crystal structure of the GlpX complex with phosphate demonstrated that the inhibitor molecule binds to the active site. Alanine replacement mutagenesis of GlpX identified 12 conserved residues important for activity and suggested that Thr 90 is the primary catalytic residue. Our data provide insight into the molecular mechanisms of the substrate specificity and catalysis of GlpX and other class II fructose-1,6-bisphosphatases.Fructose-1,6-bisphosphatase (FBPase, 2 EC 3.1.3.11), a key enzyme of gluconeogenesis, catalyzes the hydrolysis of fructose 1,6-bisphosphate to form fructose 6-phosphate and orthophosphate. It is the reverse of the reaction catalyzed by phosphofructokinase in glycolysis, and the product, fructose 6-phosphate, is an important precursor in various biosynthetic pathways (1). In all organisms, gluconeogenesis is an important metabolic pathway that allows the cells to synthesize glucose from noncarbohydrate precursors, such as organic acids, amino acids, and glycerol. FBPases are members of the large superfamily of lithium-sensitive phosphatases, which includes three families of inositol phosphatases and FBPases (the phosphoesterase clan CL0171, 3167 sequences, Pfam data base). These enzymes show metal-dependent and lithium-sensitive phosphomonoesterase activity and include inositol polyphosphate 1-phosphatases, inositol monophosphatases (IMPases), 3Ј-phosphoadenosine 5Ј-phosphatases (PAPases), and enzymes acting on both inositol 1,4-bisphosphate and PAP (PIPases) (2). They possess a common structural core with the active site lying between ␣ϩ␤ and ␣/␤ domains (3). Li ϩ -sensitive phosphatases are putative targets for lithium therapy in the treatment of manic depressive patients (4), whereas FBPases are targets f...

Brucella abortus Depends on Pyruvate Phosphate Dikinase and Malic Enzyme but Not on Fbp and GlpX Fructose-1,6-Bisphosphatases for Full Virulence in Laboratory Models

“…This is in agreement with the fact that no bacterial genome has been described as carrying a combination of classes I and III (34). Also, it has been reported that some E. coli strains carry two class II FBPases (GlpX and YggF [56]), but genomic analysis of all phosphatases in B. abortus failed to identify clear candidates for any phosphatase close to GlpX and Fbp (see Fig. S4 in the supplemental material).…”

“…The existence of a third FBPase is the first and most obvious possibility. So far, five different types of FBPases (I to V) in prokaryotes have been described (56). Whereas FBPases of classes IV and V are restricted to Archaea and their close hyperthermophilic Aquifex bacterial group, many bacteria have dual combinations of class I (Fbp homologues), class II (GlpX homologues), and class III FBPases (34,56).…”

“…So far, five different types of FBPases (I to V) in prokaryotes have been described (56). Whereas FBPases of classes IV and V are restricted to Archaea and their close hyperthermophilic Aquifex bacterial group, many bacteria have dual combinations of class I (Fbp homologues), class II (GlpX homologues), and class III FBPases (34,56). However, a genomic search for Bacillus subtilis YydE homologues (the prototype of class III FBPases [57]) in the Brucellaceae revealed only an imperfect match (a hypothetical protein of 218 amino acids with a 32% identity with the 671 amino acids in YydE) in Ochrobactrum anthropi and none in Brucella.…”

The Poor Growth of Rhodospirillum rubrum Mutants Lacking RubisCO Is Due to the Accumulation of Ribulose-1,5-Bisphosphate

“…The cycle is similar to that found in many other organisms, except no gene for sedoheptulose-1,7-bisphosphatase was found in R. rubrum. However, in many organisms the fructose-1,6-bisphosphatases, especially of the class II type, also have sedoheptulose-1,7-bisphosphatase activity (1,3,55,59,69), and a cbbF homolog in R. rubrum, Rru_A2409 (glpX), is predicted to be of this type.…”

Characterization of Fructose 1,6-Bisphosphatase and Sedoheptulose 1,7-Bisphosphatase from the Facultative Ribulose Monophosphate Cycle Methylotroph Bacillus methanolicus

“…The third block is part of the Li ϩ -sensitive phosphate motif and was shown previously by crystallographic and mutagenesis studies to be important but not sufficient for metal ion binding and catalysis (51). While FBPase II enzymes from E. coli (52), M. tuberculosis (50), Synechocystis sp. PCC6803 (25), B. subtilis (33), and C. glutamicum (42) have been biochemically characterized, the characteristics of a promiscuous FBPase/SBPase from a nonphotosynthetic bacterium lacking the Calvin cycle have not yet been determined.…”