Purification, kinetic studies, and homology model of Escherichia coli fructose-1,6-bisphosphatase

Kelley‐Loughnane, Nancy; Biolsi, Susan A; Gibson, Kate M; Lu, Guqiang; Hehir, Michael J.; Phelan, Paul J.; Kantrowitz, Evan R.

doi:10.1016/s0167-4838(01)00261-8

Cited by 27 publications

(28 citation statements)

References 36 publications

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“…E. coli possesses three FBPases, one class I FBPase, encoded by fbp (16), and two class II FBPases, encoded by glpX (19) and yggF (52). The type I FBPase, probably the main FBPase in E. coli, is essential for growth on gluconeogenic substrates (21,23) and is inhibited strongly by AMP and to a lesser extent by PEP (16,57 (19,52). As expression of glpX is induced by glycerol and glycerol 3-phosphate, GlpX is supposed to be important under these conditions rather than being active as a general FBPase under gluconeogenic conditions (58).…”

Section: Discussionmentioning

confidence: 99%

“…However, it has been shown that curing of the natural plasmid pBM19, which carries the key mdh gene and five genes with deduced roles in the RuMP cycle (glpX, fba, tkt, pfk, and rpe), resulted in the loss of the ability to grow on methanol and caused higher methanol tolerance and reduced formaldehyde tolerance (15). Transcription of mdh, all five plasmid-borne RuMP cycle genes, as well as the chromosomal genes hps and phi was increased during growth with methanol, suggesting their importance for methylotrophy (16). While pBM19 is critical for growth on methanol and is important for formaldehyde detoxification, the maintenance of this plasmid represents a burden for B. methanolicus when growing on mannitol.…”

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confidence: 99%

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

Stolzenberger¹,

Lindner

Persicke

et al. 2013

J Bacteriol

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The genome of the facultative ribulose monophosphate (RuMP) cycle methylotroph Bacillus methanolicus encodes two bisphosphatases (GlpX), one on the chromosome (GlpX C ) and one on plasmid pBM19 (GlpX P ), which is required for methylotrophy. Both enzymes were purified from recombinant Escherichia coli and were shown to be active as fructose 1,6-bisphosphatases (FBPases). The FBPase-negative Corynebacterium glutamicum ⌬fbp mutant could be phenotypically complemented with glpX C and glpX P from B. methanolicus. GlpX P and GlpX C share similar functional properties, as they were found here to be active as homotetramers in vitro, activated by Mn 2؉ ions and inhibited by Li ؉ , but differed in terms of the kinetic parameters. GlpX C showed a much higher catalytic efficiency and a lower K m for fructose 1,6-bisphosphate (86.3 s ؊1 mM ؊1 and 14 ؎ 0.5 M, respectively) than GlpX P (8.8 s ؊1 mM ؊1 and 440 ؎ 7.6 M, respectively), indicating that GlpX C is the major FBPase of B. methanolicus. Both enzymes were tested for activity as sedoheptulose 1,7-bisphosphatase (SBPase), since a SBPase variant of the ribulose monophosphate cycle has been proposed for B. methanolicus. The substrate for the SBPase reaction, sedoheptulose 1,7-bisphosphate, could be synthesized in vitro by using both fructose 1,6-bisphosphate aldolase proteins from B. methanolicus. Evidence for activity as an SBPase could be obtained for GlpX P but not for GlpX C . Based on these in vitro data, GlpX P is a promiscuous SBPase/FBPase and might function in the RuMP cycle of B. methanolicus.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Stolzenberger¹,

Lindner

Persicke

et al. 2013

J Bacteriol

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“…E. coli FBP has been shown to be activated by Mg 2ϩ and inhibited by AMP and Fru-2,6-P, similar to the mammalian enzymes, although Mg 2ϩ activation and AMP inhibition did not display cooperativity, as in the mammalian enzymes (20). The physiological relevance of Fru-2,6-P inhibition of E. coli FBP is not clear; Fru-2,6-P inhibition is not synergistic with AMP inhibition as in mammalian systems (20,21), and to date Fru-2,6-P has not been found in bacterial cells (22). Phosphoenolpyruvate has been shown to relieve AMP inhibition of E. coli FBP (21,23).…”

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confidence: 90%

Inhibition of Fructose-1,6-bisphosphatase by Aminoimidazole Carboxamide Ribotide Prevents Growth of Salmonella enterica purH Mutants on Glycerol

Dougherty¹,

Boyd

Downs

2006

Journal of Biological Chemistry

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The enzyme fructose-1,6-bisphosphatase (FBP) is key regulatory point in gluconeogenesis. Mutants of Salmonella enterica lacking purH accumulate 5-amino-4-imidazole carboxamide ribotide (AICAR) and are unable to utilize glycerol as sole carbon and energy sources. The work described here demonstrates this lack of growth is due to inhibition of FBP by AICAR. Mutant alleles of fbp that restore growth on glycerol encode proteins resistant to inhibition by AICAR and the allosteric regulator AMP. This is the first report of biochemical characterization of substitutions causing AMP resistance in a bacterial FBP. Inhibition of FBP activity by AICAR occurs at physiologically relevant concentrations and may represent a form of regulation of gluconeogenic flux in Salmonella enterica.An efficient cellular metabolism that can adapt to changing environmental conditions requires the integration of many biochemical pathways. Subtle interactions that exist between pathways in the metabolic network are in general not well understood. One level of these interactions involves metabolites common to multiple pathways. Cellular metabolites can impact physiology through roles in transcriptional regulation, translational regulation, or by mediating allosteric effects on key enzymes. One such central metabolite with regulatory capabilities is 5-amino-4-imidazole carboxamide ribotide (AICAR).

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“…Fbp is required for growth on gluconeogenic substrates and probably represents the main gluconeogenic FBPase (12). 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).…”

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confidence: 99%

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

Brown

Singer

Лунин

et al. 2009

Journal of Biological Chemistry

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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...

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Purification, kinetic studies, and homology model of Escherichia coli fructose-1,6-bisphosphatase

Cited by 27 publications

References 36 publications

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

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

Inhibition of Fructose-1,6-bisphosphatase by Aminoimidazole Carboxamide Ribotide Prevents Growth of Salmonella enterica purH Mutants on Glycerol

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

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