Mutagenesis of catalytically important residues of cupin type phosphoglucose isomerase from Archaeoglobus fulgidus

Abstract

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.

Structure-Based Annotation of a Novel Sugar Isomerase from the Pathogenic E. coli O157:H7

“…Consistent with prior studies of cupin enzymes, mutation of the metal-binding residue E110 to alanine resulted in a significant decrease in activity. 16,17,19,[29][30][31][32][33] The mutation of three residues (K108, E186, and D193) resulted in complete loss of isomerase activity. The K108 residue was found to be in contact with O2 of the fructose molecule, indicating that it may play a role in the catalytic mechanism.…”

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

“…4). 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).…”

Unusual Starch Degradation Pathway via Cyclodextrins in the Hyperthermophilic Sulfate-Reducing Archaeon Archaeoglobus fulgidus Strain 7324

“…The latter enzyme couples the conversion of acetyl-CoA to acetate to ATP generation and constitutes the mechanism of acetate formation typical for the archaeal domain (61). Several enzymes of this pathway have been purified and characterized (17,25,31,44). Thus far, the enzymes of starch degradation to intermediates of the Embden-Meyerhof pathway, i.e., glucose or glucose 6-phosphate, have not been identified in A. fulgidus.…”