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Cupin-type phosphoglucose isomerases (Cupin-PGIs) constitute a novel metal-dependent PGI family representing a convergent line of PGI evolution.

ABSTRACT: 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 Fe2+ and Ni2+, indicating a metal dependence of cPGI activity. The motifs TX3PX3GXEX3TXGHXHX6-11EXY and PPX3HX3N were deduced as the two signature patterns of the novel cPGI family. Phylogenetic analysis suggests lateral gene transfer for the bacterial cPGIs from euryarchaeota.

SUBMITTER: Hansen T

PROVIDER: S-EPMC1063998 | biostudies-literature | 2005 Mar

REPOSITORIES: biostudies-literature

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Cupin-type phosphoglucose isomerases (Cupin-PGIs) constitute a novel metal-dependent PGI family representing a convergent line of PGI evolution.

Hansen Thomas T Schlichting Bettina B Felgendreher Martina M Schönheit Peter P

Journal of bacteriology 20050301 5

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, thermophili ...[more]

PMID: 15716432

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Project description:Colias eurytheme butterflies display extensive allozyme polymorphism in the enzyme phosphoglucose isomerase (PGI). Earlier studies on biochemical and fitness effects of these genotypes found evidence of strong natural selection maintaining this polymorphism in the wild. Here we analyze the molecular features of this polymorphism by sequencing multiple alleles and modeling their structures. PGI is a dimer with rotational symmetry. Each monomer provides a critical residue to the other monomer's catalytic center. Sequenced alleles differ at multiple amino acid positions, including cryptic charge-neutral variation, but most consistent differences among the electromorph alleles are at the charge-changing amino acid sites. Principal candidate sites of selection, identified by structural and functional analyses and by their variants' population frequencies, occur in interpenetrating loops across the interface between monomers, where they may alter subunit interactions and catalytic center geometry. Comparison to a second (and basal) species, Colias meadii, also polymorphic for PGI under natural selection, reveals one fixed amino acid difference between their PGIs, which is located in the interpenetrating loop and accompanies functional differences among their variants. We also study nucleotide variability among the PGI alleles, comparing these data to similar data from another glycolytic enzyme gene, glyceraldehyde-3-phosphate dehydrogenase. Despite extensive nonsynonymous and synonymous polymorphism at PGI in each species, the only base changes fixed between species are the two causing the amino acid replacement; this absence of synonymous fixation yields a significant McDonald-Kreitman test. Analyses of these data suggest historical population expansion. Positive peaks of Tajima's D statistic, representing regions of neutral "hitchhiking," are found around the principal candidate sites of selection. This study provides novel views of molecular-structural mechanisms, and beginnings of historical evidence, for a long-persistent balanced enzyme polymorphism at PGI in these and perhaps other species.

Project description:Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth. Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction. However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking. Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily. EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure. The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme. We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency. Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants. An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel.

Dataset Information

Cupin-type phosphoglucose isomerases (Cupin-PGIs) constitute a novel metal-dependent PGI family representing a convergent line of PGI evolution.

Publications

Cupin-type phosphoglucose isomerases (Cupin-PGIs) constitute a novel metal-dependent PGI family representing a convergent line of PGI evolution.

Similar Datasets

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