The crystal structure of human phosphoglucose isomerase at 1.6 Å resolution: implications for catalytic mechanism, cytokine activity and haemolytic anaemia

Glycerolipids are structural components for membranes and serve in energy storage. We describe here the use of a photodynamic selection technique to generate a population of Chinese hamster ovary cells that display a global deficiency in glycerolipid biosynthesis. One isolate from this population, GroD1, displayed a profound reduction in the synthesis of phosphatidylcholine, phosphatidylethanolamine, and triglycerides but presented high levels of phosphatidic acid and normal levels of phosphatidylinositol synthesis. This was accompanied by a reduction in phosphatidate phosphatase 1 (PAP1) activity. Expression cloning and sequencing of the cDNA obtained from GroD1 revealed a point mutation, Gly-189 3 Glu, in glucose-6-phosphate isomerase (GPI), a glycolytic enzyme involved in an inherited disorder that results in anemia and neuromuscular symptoms in humans. GPI activity was reduced by 87% in GroD1. No significant differences were found in DNA synthesis, protein synthesis, and ATP levels, whereas glycerol 3-phosphate levels were increased in the mutant. Expression of wild-type hamster GPI restored GPI activity, glycerolipid biosynthesis, and PAP1 activity in GroD1. Two additional, independently isolated GPI-deficient mutants displayed similar phenotypes with respect to PAP1 activity and glycerolipid biosynthesis. These findings uncover a novel relationship between GPI, involved in carbohydrate metabolism, and PAP1, a lipogenic enzyme. These results may also help to explain neuromuscular symptoms associated with inherited GPI deficiency.

Section: Expression Cloning Reveals a Mutation In Glucose-6-phosphatementioning

confidence: 99%

Isolation of Novel Animal Cell Lines Defective in Glycerolipid Biosynthesis Reveals Mutations in Glucose-6-phosphate Isomerase

Haller

Smith

Liu

et al. 2010

“…PGI structures have been solved from a variety of mammalian sources and from B. stearothermophilus in native, inhibitor, and substrate (F6P)-bound forms (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). CD spectra analysis and secondary structure predictions suggest a similar fold of the PGI/PMI from A. pernix and T. acidophilum and e.g.…”

Section: Molecular and Thermophilic Properties-thementioning

confidence: 99%

Bifunctional Phosphoglucose/Phosphomannose Isomerases from the Archaea Aeropyrum pernix and Thermoplasma acidophilum Constitute a Novel Enzyme Family within the Phosphoglucose Isomerase Superfamily

Hansen

Wendorff

Schönheit

2004

The hyperthermophilic crenarchaeon Aeropyrum pernix contains phosphoglucose isomerase (PGI) activity. However, obvious homologs with significant identity to known PGIs could not be identified in the sequenced genome of this organism. The PGI activity from A. pernix was purified and characterized. Kinetic analysis revealed that, unlike all known PGIs, the enzyme catalyzed reversible isomerization not only of glucose 6-phosphate but also of epimeric mannose 6-phosphate at similar catalytic efficiency, thus defining the protein as bifunctional phosphoglucose/phosphomannose isomerase (PGI/PMI). The gene pgi/pmi encoding PGI/PMI (open reading frame APE0768) was identified by matrixassisted laser desorption ionization time-of-flight analyses; the gene was overexpressed in Escherichia coli as functional PGI/PMI. Putative PGI/PMI homologs were identified in several (hyper)thermophilic archaea and two bacteria. The homolog from Thermoplasma acidophilum (Ta1419) was overexpressed in E. coli, and the recombinant enzyme was characterized as bifunctional PGI/PMI. PGI/PMIs showed low sequence identity to the PGI superfamily and formed a distinct phylogenetic cluster. However, secondary structure predictions and the presence of several conserved amino acids potentially involved in catalysis indicate some structural and functional similarity to the PGI superfamily. Thus, we propose that bifunctional PGI/PMI constitutes a novel protein family within the PGI superfamily.Phosphoglucose isomerase (PGI 1 ; EC 5.3.1.9) catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate. PGI plays a central role in sugar metabolism of eukarya, bacteria, and Archaea both in glycolysis via the Embden-Meyerhof pathway in eukarya and bacteria and in its modified versions found in Archaea. PGI is also involved in gluconeogenesis where the enzyme operates in the reverse direction (for the literature see Refs. 1-3). PGIs from the domains of eukarya and bacteria are well studied enzymes. A variety of PGIs have been purified and biochemically characterized, and the encoding genes have been cloned and sequenced (e.g. Refs. 4 -11). Crystal structures have been determined for the eukaryotic PGIs from pig, rabbit, human, and from the bacterium Bacillus stearothermophilus, and conserved amino acids proposed to be involved in substrate binding and/or catalysis have been identified (12-15, 17, 18, 20 -25). The eukaryal and bacterial PGIs belong to the PGI superfamily defined by its two conserved signature patternsTo date this superfamily includes more than 300 PGI sequences (see www.sanger.ac.uk/cgi-bin/Pfam/getacc?PF00342) (26) from bacteria and eukarya but only three from the archaeal domain. These include the PGI from the hyperthermophilic euryarchaeon Methanococcus jannaschii (MjPGI). This PGI has recently been characterized as the first archaeal member of the PGI superfamily. 2 So far little is known about other archaeal PGIs. Recently, two other euryarchaeal PGIs have been characterized, one from the hyperthermophilic euryarch...

“…Conformational Changes upon Ligand Binding-In conventional PGI, a number of conformational changes associated with ligand binding have been described (9,26,27). These comprise a domain closure around the phosphate group and two helix movements that position a histidine and a lysine residue, respectively, both of which likely have a role in ring opening.…”

Section: Figmentioning

confidence: 99%

Structural Evidence for a Hydride Transfer Mechanism of Catalysis in Phosphoglucose Isomerase from Pyrococcus furiosus

Swan

Solomons²,

Beeson

et al. 2003

Self Cite

In the Euryarchaeota species Pyrococcus furiosus and Thermococcus litoralis, phosphoglucose isomerase (PGI) activity is catalyzed by an enzyme unrelated to the well known family of PGI enzymes found in prokaryotes, eukaryotes, and some archaea. We have determined the crystal structure of PGI from Pyrococcus furiosus in native form and in complex with two active site ligands, 5-phosphoarabinonate and gluconate 6-phosphate. In these structures, the metal ion, which in vivo is presumed to be Fe 2؉ , is located in the core of the cupin fold and is immediately adjacent to the C1-C2 region of the ligands, suggesting that Fe 2؉ is involved in catalysis rather than serving a structural role. The active site contains a glutamate residue that contacts the substrate, but, because it is also coordinated to the metal ion, it is highly unlikely to mediate proton transfer in a cis-enediol mechanism. Consequently, we propose a hydride shift mechanism of catalysis. In this mechanism, Fe 2؉ is responsible for proton transfer between O1 and O2, and the hydride shift between C1 and C2 is favored by a markedly hydrophobic environment in the active site. The absence of any obvious enzymatic machinery for catalyzing ring opening of the sugar substrates suggests that pyrococcal PGI has a preference for straight chain substrates and that metabolism in extreme thermophiles may use sugars in both ring and straight chain forms.Phosphoglucose isomerase (PGI) 1 (E.C. 5.3.1.9) catalyzes the interconversion of glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P), which are substrates of glycolysis and gluconeogenesis. The enzyme is represented by two evolutionarily distinct protein families. In eubacteria, eukaryotes, and a few archaea, the enzyme is of the conventional type that has been studied extensively at the biochemical and structural level. To date, the second, novel type has been found in two Euryarchaeota species, Pyrococcus furiosus and Thermococcus litoralis (1-3). This is markedly smaller than conventional PGI; for instance, the enzyme from P. furiosus comprises a dimer of 43 kDa (1, 2), whereas mammalian PGI is a 132-kDa dimer. Based on sequence alignments, it has been suggested that the novel type of PGI contains a cupin fold (2), and this has been confirmed by a recent crystal structure of the enzyme from Pyrococcus furiosus (4). The hallmark of this fold is a ␤ barrel-like structure that frequently, but not exclusively, contains a metal-binding site (for review, see Ref. 5). PGI from T. litoralis, which is highly similar to the enzyme from P. furiosus, contains iron with traces of zinc (3).The reaction catalyzed by PGI is an aldose-ketose isomerization in which a hydrogen atom is transferred between the C1 and C2 positions of the substrate. A second hydrogen, in the form of a proton, also moves between the O1 and O2. The carbon-bound hydrogen can move by one of two mechanisms, a hydride shift or a proton transfer via a cis-enediol intermediate (6). In isomerases that contain a metal ion at the catalytic center the mechanis...