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

Abstract

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...

Cupin-Type Phosphoglucose Isomerases (Cupin-PGIs) Constitute a Novel Metal-Dependent PGI Family Representing a Convergent Line of PGI Evolution

“…cPGIs show neither homology (ca. 5%) nor structural similarity to the PGI superfamily and represent a convergent line of PGI evolution (4,22,49,52). Their function as cPGIs has been described either recently (22,30,52) or in the present study.…”

“…Thus, the data presented here, and in particular the reactivation experiments, indicate an involvement of metals in catalysis. Moreover, the presence of a divalent metal, most likely iron, as deduced from the crystal structure of PfcPGI bound with 6-phosphogluconate is a prerequisite in the recently proposed hydride transfer mechanism of PfcPGI (49).…”

“…Sequence comparison suggested the presence of few putative cPGI homologs; however, none of them have been characterized (44, 53). Two consensus sequences (GX 5 HXHX 3,4 EX 6 G and GX 5 PXGX 2 HX 3 N) were established for the cupin superfamily (17, 18), which include a metal-binding site found in most cupin enzymes, including the PfcPGI and TlPGI (4,30,49). Two crystal structures are available for the PfcPGI; however, the role of the metal is discussed to be either involved in substrate binding and/or in catalysis (4,30,49).…”

“…Two consensus sequences (GX 5 HXHX 3,4 EX 6 G and GX 5 PXGX 2 HX 3 N) were established for the cupin superfamily (17, 18), which include a metal-binding site found in most cupin enzymes, including the PfcPGI and TlPGI (4,30,49). Two crystal structures are available for the PfcPGI; however, the role of the metal is discussed to be either involved in substrate binding and/or in catalysis (4,30,49). Berrisford et al (4) showed that catalytic activity is not strongly influenced by the presence or absence of the bound metal, suggesting that the metal might be implicated in substrate recognition rather than being directly involved in catalysis.…”

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

“…In contrast to conventional PGIs, cPGI activity is strictly dependent on bivalent metal ions such as Fe 2ϩ and Ni 2ϩ (74,93). Two different catalytic mechanisms, proceeding via (i) a carbocation and a subsequent hydride shift and (ii) a cis-enediolate intermediate with a conserved glutamate residue (E97) as a base catalyst, as described above, have been proposed (94)(95)(96). In both proposed mechanisms, the metal dependence is explained by the coordination of the substrate in the active site not being involved directly in catalysis (95).…”