Spontaneous subunit exchange in porcine liver fructose‐1,6‐bisphosphatase

Nelson, Scott W.; Honzatko, Richard B.; Fromm, Herbert J.

doi:10.1016/s0014-5793(01)02262-1

Cited by 15 publications

(16 citation statements)

References 38 publications

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“…The Hill coefficients for both Fru-1,6-P 2 and Mg 2ϩ are 1.1 Ϯ 0.1. These values are similar to those reported previously for E. coli FBPase (4, 14, 16), and differ from the porcine enzyme principally in the Hill coefficient for Mg 2ϩ , which for porcine FBPase is 1.9 Ϯ 0.1 (45).…”

Section: Resultssupporting

confidence: 90%

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Novel Allosteric Activation Site in Escherichia coli Fructose-1,6-bisphosphatase

Hines

Fromm

Honzatko

2006

Journal of Biological Chemistry

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Fructose-1,6-bisphosphatase (FBPase) governs a key step in gluconeogenesis, the conversion of fructose 1,6-bisphosphate into fructose 6-phosphate. In mammals, the enzyme is subject to metabolic regulation, but regulatory mechanisms of bacterial FBPases are not well understood. Presented here is the crystal structure (resolution, 1.45 Å ) of recombinant FBPase from Escherichia coli, the first structure of a prokaryotic Type I FBPase. The E. coli enzyme is a homotetramer, but in a quaternary state between the canonical Rand T-states of porcine FBPase. Phe 15 and residues at the C-terminal side of the first ␣-helix (helix H1) occupy the AMP binding pocket. Residues at the N-terminal side of helix H1 hydrogen bond with sulfate ions buried at a subunit interface, which in porcine FBPase undergoes significant conformational change in response to allosteric effectors. Phosphoenolpyruvate and sulfate activate E. coli FBPase by at least 300%. Key residues that bind sulfate anions are conserved among many heterotrophic bacteria, but are absent in FBPases of organisms that employ fructose 2,6-bisphosphate as a regulator. These observations suggest a new mechanism of regulation in the FBPase enzyme family: anionic ligands, most likely phosphoenolpyruvate, bind to allosteric activator sites, which in turn stabilize a tetramer and a polypeptide fold that obstructs AMP binding.Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3. 1.3.11; FBPase) 2 is a key enzyme of the gluconeogenic pathway, hydrolyzing fructose 1,6-bisphosphate (Fru-1,6-P 2 ) to fructose 6-phosphate (Fru-6-P) and inorganic phosphate (P i ) (1, 2). In Escherichia coli, the most likely role of FBPase is the generation of fivecarbon precursors for nucleotide and polysaccharide production (3, 4). Microbial strains without FBPase (gene deletion mutants) cannot grow on gluconeogenic substrates, but thrive on glucose or other hexoses (3).FBPase and fructose-6-phosphate-1-kinase are constitutive enzymes in E. coli, and in the absence of some kind of coordinate regulation, define a futile cycle (5). Futile cycling, however, is minimal under glycolytic (6) and gluconeogenic (7) conditions, and hence FBPase must be under metabolic control. In mammals, AMP and fructose 2,6-bisphosphate (Fru-2,6-P 2 ) synergistically inhibit FBPase (8). Fru-2,6-P 2 , the concentration of which is subject to hormonal control, directly inhibits FBPase by binding to its active site, whereas AMP binds to an allosteric site (5, 9, 10) and induces a conformational change, converting the enzyme from an active quaternary conformation (R-state) to an inactive form (T-state) (11-13). In contrast, little is known regarding the regulation of FBPase in E. coli. AMP is a potent inhibitor of the E. coli enzyme (4, 14 -16), but Fru-2,6-P 2 is not present. (Fru-2,6-P 2 , however, does inhibit E. coli FBPase in assays (15, 16).) Dynamic regulation by AMP alone is unlikely, because AMP concentrations in vivo remain relatively constant under gluconeogenic and glycolytic condit...

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Section: Resultssupporting

confidence: 90%

“…Porcine FBPase undergoes spontaneous subunit exchange (45,52,53). In the presence of Fru-1,6-P 2 or Fru-2,6-P 2 , exchange reactions involve only dimers of the tetramer (45,53).…”

Section: Discussionmentioning

confidence: 99%

Novel Allosteric Activation Site in Escherichia coli Fructose-1,6-bisphosphatase

Hines

Fromm

Honzatko

2006

Journal of Biological Chemistry

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“…2. In the presence of F16P 2 , subunit exchange occurs only at the level of C1/C2 dimers (28). Hence, protocol B leads to hybrids 4:0, 0:4, and 2:2p.…”

Section: Methodsmentioning

confidence: 99%

“…Anion-exchange chromatography cleanly separates most of the hybrid constructs (28). More recently, Kantrowitz and colleagues (29) reported that AMP must bind to two subunits of FBPase to cause significant inhibition.…”

mentioning

confidence: 99%

Hybrid Tetramers of Porcine Liver Fructose-1,6-bisphosphatase Reveal Multiple Pathways of Allosteric Inhibition

Nelson

Honzatko

Fromm

2002

Journal of Biological Chemistry

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Fructose-1,6-bisphosphatase is a square planar tetramer of identical subunits, which exhibits cooperative allosteric inhibition of catalysis by AMP. Protocols for in vitro subunit exchange provide three of five possible hybrid tetramers of fructose-1,6-bisphosphatase in high purity. The two hybrid types with different subunits in the top and bottom halves of the tetramer co-purify. Hybrid tetramers, formed from subunits unable to bind AMP and subunits with wild-type properties, differ from the wild-type enzyme only in regard to their properties of AMP inhibition. Hybrid tetramers exhibit cooperative, potent, and complete (100%) AMP inhibition if at least one functional AMP binding site exists in the top and bottom halves of the tetramer. Furthermore, titrations of hybrid tetramers with AMP, monitored by a tryptophan reporter group, reveal cooperativity and fluorescence changes consistent with an R-to T-state transition, provided that again at least one functional AMP site exists in the top and bottom halves of the tetramer. In contrast, hybrid tetramers, which have functional AMP binding sites in only one half (top/bottom), exhibit an R-to T-state transition and complete AMP inhibition, but without cooperativity. Evidently, two pathways of allosteric inhibition of fructose-1,6-bisphosphatase are possible, only one of which is cooperative.

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“…Since the human muscle and liver FBPase isoforms share only 76.9% sequence identity (89.3% homology) over 337 residues and the muscle isoform has additional functions in the cell, including higher sensitivity of hmFBPase to AMP and its regulation by Ca 2+ (Gizak et al, 2012;Pirog et al, 2014), some differences in their R/T transition behaviour might be expected. In solution, porcine FBPase subunits have been shown to exchange on a time scale of a few hours (Nelson et al, 2001), indicating flexibility at the interfaces. Thus, the possibly that the rotation angle of FBPases might sometimes be influenced by crystal-packing effects cannot be discounted entirely.…”

Section: Figurementioning

confidence: 99%

Quadruple space-group ambiguity owing to rotational and translational noncrystallographic symmetry in human liver fructose-1,6-bisphosphatase

Ruf

Tetaz

Schott

et al. 2016

Acta Cryst Sect D Struct Biol

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Fructose-1,6-bisphosphatase (FBPase) is a key regulator of gluconeogenesis and a potential drug target for type 2 diabetes. FBPase is a homotetramer of 222 symmetry with a major and a minor dimer interface. The dimers connected via the minor interface can rotate with respect to each other, leading to the inactive T-state and active R-state conformations of FBPase. Here, the first crystal structure of human liver FBPase in the R-state conformation is presented, determined at a resolution of 2.2 Å in a tetragonal setting that exhibits an unusual arrangement of noncrystallographic symmetry (NCS) elements. SelfPatterson function analysis and various intensity statistics revealed the presence of pseudo-translation and the absence of twinning. The space group is P4 1 2 1 2, but structure determination was also possible in space groups P4 3 2 1 2, P4 1 22 and P4 3 22. All solutions have the same arrangement of three C 2 -symmetric dimers spaced by 1/3 along an NCS axis parallel to the c axis located at (1/4, 1/4, z), which is therefore invisible in a self-rotation function analysis. The solutions in the four space groups are related to one another and emulate a body-centred lattice. If all NCS elements were crystallographic, the space group would be I4 1 22 with a c axis three times shorter and a single FBPase subunit in the asymmetric unit. I4 1 22 is a minimal, non-isomorphic supergroup of the four primitive tetragonal space groups, explaining the space-group ambiguity for this crystal.

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Spontaneous subunit exchange in porcine liver fructose‐1,6‐bisphosphatase

Cited by 15 publications

References 38 publications

Novel Allosteric Activation Site in Escherichia coli Fructose-1,6-bisphosphatase

Novel Allosteric Activation Site in Escherichia coli Fructose-1,6-bisphosphatase

Hybrid Tetramers of Porcine Liver Fructose-1,6-bisphosphatase Reveal Multiple Pathways of Allosteric Inhibition

Quadruple space-group ambiguity owing to rotational and translational noncrystallographic symmetry in human liver fructose-1,6-bisphosphatase

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