Cupin-type phosphoglucose isomerases (cPGIs) were identified in some archaeal and bacterial genomes and the respective coding function of cpgi's from the euryarchaeota Archaeoglobus fulgidus and Methanosarcina mazei, as well as the bacteria Salmonella enterica serovar Typhimurium and Ensifer meliloti, was proven by functional overexpression. These cPGIs and the cPGIs from Pyrococcus and Thermococcus spp. represent the cPGI family and were compared with respect to kinetic, inhibitory, thermophilic, and metal-binding properties. cPGIs showed a high specificity for the substrates fructose-6-phosphate and glucose-6-phosphate and were inhibited by millimolar concentrations of sorbitol-6-phosphate, erythrose-4-phosphate, and 6-phosphogluconate. Treatment of cPGIs with EDTA resulted in a complete loss of catalytic activity, which could be regained by the addition of some divalent cations, most effectively by Fe 2؉ and Ni 2؉ , indicating a metal dependence of cPGI activity. The motifs TX 3 PX 3 GXEX 3 TXGHXHX 6-11 EXY and PPX 3 HX 3 N were deduced as the two signature patterns of the novel cPGI family. Phylogenetic analysis suggests lateral gene transfer for the bacterial cPGIs from euryarchaeota.Phosphoglucose isomerase (PGI; 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 (see references 22, 26, and 40). PGIs have evolved convergently. Most PGIs belong to the PGI superfamily, which can be divided into the PGI family and the recently identified bifunctional phosphoglucose/phosphomannose isomerase (PGI/PMI) family (22). PGIs from the PGI superfamily, often referred to as conventional PGIs, are found in all domains of life and are well studied. Crystal structures have been determined for the eukaryotic PGIs from pigs, rabbits, humans and from the bacterium Bacillus stearothermophilus, and conserved amino acids proposed to be involved in substrate binding and/ or catalysis have been identified (3, 6, 10-13, 28, 29, 42). Bifunctional PGI/PMIs, which have been characterized as a novel family within the PGI superfamily, were predominantly found in the crenarchaeotal branch of the archaea (26).Recently, a novel type of PGI has been identified and characterized from the hyperthermophilic euryarchaeon Pyrococcus furiosus (22, 52) and later from the closely related Thermococcus litoralis (30). These PGIs belong to the cupin superfamily and thus represent a convergent line of PGI evolution. The cupin superfamily is present in all three domains of life-eukarya, bacteria, and archaea-and comprises a group of functionally diverse proteins that contain a central domain composed of ␤-strands forming a small ␤-barrel called "cupin."Proteins from the cupin superfamily range, for example, from manno...

The gene (open reading frame Tm1155, g6pd) encoding glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) of the hyperthermophilic bacterium Thermotoga maritima was cloned and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 95 kDa composed of 60-kDa subunits. Rate dependence (at 80 degrees C) on glucose-6-phosphate and NADP(+) followed Michaelis-Menten kinetics with apparent K(m) values of 0.15 mM and 0.03 mM, respectively; apparent V(max) values were about 20 U mg(-1). The enzyme also reduced NAD(+) (apparent K(m) 12 mM, V(max) 12 U mg(-1)). The 1000-fold higher catalytic activity (k(cat)/K(m)) with NADP(+) over NAD(+) defines the G6PD as NADP(+) specific in vivo. G6PD activity was competitively inhibited by NADPH with a K(i) value of 0.11 mM. With a temperature optimum of 92 degrees C the enzyme is the most thermoactive G6PD described.

Phosphoglucose isomerase (PGI; EC 5.3.1.9) catalyzes the reversible aldose-ketose 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 the modified versions of this pathway found in archaea. PGI is also involved in gluconeogenesis, where the enzyme operates in the reverse direction [1][2][3]. Two lines of PGIs have evolved independently: the PGI superfamily and the cupin type PGIs (cPGIs) [2,4,5]. The PGI superfamily, which includes the vast majority of known PGIs, has recently been divided into the PGI family, which is found in almost all bacteria and eukarya, and the PGI ⁄ PMI-family, which is predominantly found in crenarchaeota [5]. A cupin type PGI was first described for Pyrococcus furiosus (PfcPGI) [2,6]. To date, 14 members of this novel family, which is presumably of Recently, cupin type phosphoglucose isomerases have been described as a novel protein family representing a separate lineage in the evolution of phosphoglucose isomerases. The importance of eight active site residues completely conserved within the cPGI family has been assessed by sitedirected mutagenesis using the cPGI from Archaeoglobus fulgidus (AfcPGI) as a model. The mutants T63A, G79A, G79L, H80A, H80D, H82A, E93A, E93D, Y95F, Y95K, H136A, and Y160F were constructed, purified, and the impact of the respective mutation on catalysis and ⁄ or metal ion binding as well as thermostability was analyzed. The variants G79A, G79L, and Y95F exhibited a lower thermostability. The catalytic efficiency of the enzyme was reduced by more than 100-fold in the G79A, G79L, H80A, H80D, E93D, Y95F variants and more than 15-fold in the T63A, H82A, Y95K, Y160F variants, but remained about the same in the H136A variant at Ni 2+ saturating conditions. Further, the Ni 2+ content of the mutants H80A, H80D, H82A, E93A, E93D and their apparent Ni 2+ binding ability was reduced, resulting in an almost complete loss of activity and thus underlining the crucial role of the metal ion for catalysis. Evidence is presented that H80, H82 and E93 play an additional role in catalysis besides metal ion binding. E93 appears to be the key catalytic residue of AfcPGI, as the E93A mutant did not show any catalytic activity at all.Abbreviations 6-PG, 6-phosphogluconate; AfcPGI, cPGI from Archaeoglobus fulgidus; cPGI, cupin type phosphoglucose isomerase; EmcPGI1, cPGI isoenzyme 1 from Ensifer meliloti; EmcPGI2, cPGI isoenzyme 2 from Ensifer meliloti; F6P, fructose-6-phosphate; G6P, glucose-6-phosphate; MbucPGI, Methanococcoides burtonii; MtcPGI, cPGI from Moorella thermoacetica; PfcPGI, cPGI from Pyrococcus furiosus; PGI, conventional phosphoglucose isomerase; PGI ⁄ PMI, phosphoglucose isomerase ⁄ phosphomannose isomerase; StcPGI, cPGI from Salmonella typhimurium; TkcPGI, cPGI from Thermococcus kodakaraensis; TlcPGI, cPGI from Thermococcus litoralis.