Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.1.2.13 (aldolase)
3,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the reversible cleavage of KDPG to pyruvate and glyceraldehyde-3-phosphate. The enzyme is a class I aldolase whose reaction mechanism involves formation of Schiff base intermediates between Lys-133 and a keto substrate. A covalent adduct was trapped by flash freezing KDPG aldolase crystals soaked with 10 mM pyruvate in acidic conditions at pH 4.6. Structure determination to 1.95-A resolution showed that pyruvate had undergone nucleophilic attack with Lys-133, forming a protonated carbinolamine intermediate, a functional Schiff base precursor, which was stabilized by hydrogen bonding with active site residues. Carbinolamine interaction with Glu-45 indicates general base catalysis of several rate steps. Stereospecific addition is ensured by aromatic interaction of Phe-135 with the pyruvate methyl group. In the native structure, Lys-133 donates all of its hydrogen bonds, indicating the presence of an epsilon-ammonium salt group. Nucleophilic activation is postulated to occur by proton transfer in the monoprotonated zwitterionic pair (Glu-45/Lys-133). Formation of the zwitterionic pair requires prior side chain rearrangement by protonated Lys-133 to displace a water molecule, hydrogen bonded to the zwitterionic residues.
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PMID:Covalent intermediate trapped in 2-keto-3-deoxy-6- phosphogluconate (KDPG) aldolase structure at 1.95-A resolution. 1127 85

Fructose-1,6-bis(phosphate) aldolase is an essential glycolytic enzyme found in all vertebrates and higher plants that catalyzes the cleavage of fructose 1,6-bis(phosphate) (Fru-1,6-P(2)) to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). Mutations in the aldolase genes in humans cause hemolytic anemia and hereditary fructose intolerance. The structure of the aldolase-DHAP Schiff base has been determined by X-ray crystallography to 2.6 A resolution (R(cryst) = 0.213, R(free) = 0.249) by trapping the catalytic intermediate with NaBH(4) in the presence of Fru-1,6-P(2). This is the first structure of a trapped covalent intermediate for this essential glycolytic enzyme. The structure allows the elucidation of a comprehensive catalytic mechanism and identification of a conserved chemical motif in Schiff-base aldolases. The position of the bound DHAP relative to Asp33 is consistent with a role for Asp33 in deprotonation of the C4-hydroxyl leading to C-C bond cleavage. The methyl side chain of Ala31 is positioned directly opposite the C3-hydroxyl, sterically favoring the S-configuration of the substrate at this carbon. The "trigger" residue Arg303, which binds the substrate C6-phosphate group, is a ligand to the phosphate group of DHAP. The observed movement of the ligand between substrate and product phosphates may provide a structural link between the substrate cleavage and the conformational change in the C-terminus associated with product release. The position of Glu187 in relation to the DHAP Schiff base is consistent with a role for the residue in protonation of the hydroxyl group of the carbinolamine in the dehydration step, catalyzing Schiff-base formation. The overlay of the aldolase-DHAP structure with that of the covalent enzyme-dihydroxyacetone structure of the mechanistically similar transaldolase and KDPG aldolase allows the identification of a conserved Lys-Glu dyad involved in Schiff-base formation and breakdown. The overlay highlights the fact that Lys146 in aldolase is replaced in transaldolase with Asn35. The substitution in transaldolase stabilizes the enamine intermediate required for the attack of the second aldose substrate, changing the chemistry from aldolase to transaldolase.
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PMID:Snapshots of catalysis: the structure of fructose-1,6-(bis)phosphate aldolase covalently bound to the substrate dihydroxyacetone phosphate. 1170 76

Growth of Salinibacter ruber, a red, extremely halophilic bacterium phylogenetically affiliated with the Flavobacterium/Cytophaga branch of the domain Bacteria, is stimulated by a small number of sugars (glucose, maltose, starch at 1 g l(-1)). Glucose consumption starts after other substrates have been depleted. Glucose metabolism proceeds via a constitutive, salt-inhibited hexokinase and a constitutive salt-dependent nicotinamide adenine dinucleotide phosphate (NADP)-linked glucose-6-phosphate dehydrogenase. Glucose dehydrogenase and fructose-1,6-bisphosphate aldolase activity could not be detected. It is therefore suggested that Salinibacter metabolizes glucose by the classic Entner-Doudoroff pathway and not by the Embden-Meyerhof glycolytic pathway or by the modified Entner-Doudoroff pathway present in halophilic Archaea of the family Halobacteriaceae, in which the phosphorylation step is postponed. However, activity of 2-keto-3-deoxy-6-phosphogluconate aldolase could not be detected in extracts of Salinibacter cells, whether or not grown in the presence of glucose.
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PMID:Sugar metabolism in the extremely halophilic bacterium Salinibacter ruber. 1279 4

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from Pseudomonas putida is a key enzyme in the Entner-Doudoroff pathway which catalyses the cleavage of KDPG via a class I Schiff-base mechanism. The crystal structure of this enzyme has been refined to a crystallographic residual R = 17.1% (R(free) = 21.4%). The N-terminal helix caps one side of the torus of the (betaalpha)(8)-barrel and the active site is located on the opposite, carboxylic side of the barrel. The Schiff-base-forming Lys145 is coordinated by a sulfate (or phosphate) ion and two solvent water molecules. The interactions that stabilize the trimer are predominantly hydrophobic, with the exception of the cyclically permuted bonds formed between Glu132 OE1 of one molecule and Thr129 OG1 of a symmetry-equivalent molecule. Except for the N-terminal helix, the structure of KDPG aldolase from P. putida closely resembles the structure of the homologous enzyme from Escherichia coli.
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PMID:Structure of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from Pseudomonas putida. 1287 49

A novel bacterial in vivo selection for pyruvate aldolase activity is described. Pyruvate kinase deficient cells, which lack the ability to biosynthetically generate pyruvate, require supplementation of exogenous pyruvate when grown on ribose. Supplementation with pyruvate concentrations as low as 50 microM rescues cell growth. A known substrate of the KDPG aldolases, 2-keto-4-hydroxy-4-(2'-pyridyl)butyrate (KHPB), also rescues cell growth, consistent with retroaldol cleavage by KDPG aldolase and rescue through pyruvate release. An initial round of selection against 2-keto-4-hydroxyoctonate (KHO), a nonsubstrate for wild-type aldolase, produced three mutants with intriguing alterations in protein sequence. This selection system allows rapid screening of mutant enzyme libraries and facilitates the discovery of enzymes with novel substrate specificities.
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PMID:A bacterial selection for the directed evolution of pyruvate aldolases. 1524 84

In vivo, 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the reversible, stereospecific retro-aldol cleavage of KDPG to pyruvate and D-glyceraldehyde-3-phosphate. The enzyme is a lysine-dependent (Class I) aldolase that functions through the intermediacy of a Schiff base. Here, we propose a mechanism for this enzyme based on crystallographic studies of wild-type and mutant aldolases. The three dimensional structure of KDPG aldolase from the thermophile Thermotoga maritima was determined to 1.9A. The structure is the standard alpha/beta barrel observed for all Class I aldolases. At the active site Lys we observe clear density for a pyruvate Schiff base. Density for a sulfate ion bound in a conserved cluster of residues close to the Schiff base is also observed. We have also determined the structure of a mutant of Escherichia coli KDPG aldolase in which the proposed general acid/base catalyst has been removed (E45N). One subunit of the trimer contains density suggesting a trapped pyruvate carbinolamine intermediate. All three subunits contain a phosphate ion bound in a location effectively identical to that of the sulfate ion bound in the T. maritima enzyme. The sulfate and phosphate ions experimentally locate the putative phosphate binding site of the aldolase and, together with the position of the bound pyruvate, facilitate construction of a model for the full-length KDPG substrate complex. The model requires only minimal positional adjustments of the experimentally determined covalent intermediate and bound anion to accommodate full-length substrate. The model identifies the key catalytic residues of the protein and suggests important roles for two observable water molecules. The first water molecule remains bound to the enzyme during the entire catalytic cycle, shuttling protons between the catalytic glutamate and the substrate. The second water molecule arises from dehydration of the carbinolamine and serves as the nucleophilic water during hydrolysis of the enzyme-product Schiff base. The second water molecule may also mediate the base-catalyzed enolization required to form the carbon nucleophile, again bridging to the catalytic glutamate. Many aspects of this mechanism are observed in other Class I aldolases and suggest a mechanistically and, perhaps, evolutionarily related family of aldolases distinct from the N-acetylneuraminate lyase (NAL) family.
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PMID:Mechanism of the Class I KDPG aldolase. 1640 39

Enzymes that utilize a Schiff-base intermediate formed with their substrates and that share the same alpha/beta barrel fold comprise a mechanistically diverse superfamily defined in the SCOPS database as the class I aldolase family. The family includes the "classical" aldolases fructose-1,6-(bis)phosphate (FBP) aldolase, transaldolase, and 2-keto-3-deoxy-6-phosphogluconate aldolase. Moreover, the N-acetylneuraminate lyase family has been included in the class I aldolase family on the basis of similar Schiff-base chemistry and fold. Herein, we generate primary sequence identities based on structural alignment that support the homology and reveal additional mechanistic similarities beyond the common use of a lysine for Schiff-base formation. The structural and mechanistic correspondence comprises the use of a catalytic dyad, wherein a general acid/base residue (Glu, Tyr, or His) involved in Schiff-base chemistry is stationed on beta-strand 5 of the alpha/beta barrel. The role of the acid/base residue was probed by site-directed mutagenesis and steady-state and pre-steady-state kinetics on a representative member of this family, FBP aldolase. The kinetic results are consistent with the participation of this conserved residue or position in the protonation of the carbinolamine intermediate and dehydration of the Schiff base in FBP aldolase and, by analogy, the class I aldolase family.
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PMID:New superfamily members identified for Schiff-base enzymes based on verification of catalytically essential residues. 1683 28

Changes in functional activity of specific enzyme reactions in the cells of pectinolytic bacteria from the gastrointestinal tract of animals in vitro cultivated in the medium containing pectin or glucose were studied against a background of the low dose effect of the wide spread biocide pentachlorophenol alone as well as in combination with the natural sorbents clinoptilolites. Regardless of the absence of transketolase reaction in the cells of all studied strains, they metabolized highly the above substrates that are dissimilar in chemical structure and produced different products of their degradation. It has been shown that the high metabolic level in the cells is provided by the function of the unique enzymatic reaction catalyzed by 2-keto-3-desoxy-6-phosphogluconate aldolase (EC 4.1.2.14) that permits to use effectively the metabolic pathway of Entner-Doudoroff. Cells could also utilize the same substrates via the Embden-Meyerhof-Parnas pathway, therefore they possess the other key reaction that is catalyzed by fructosobiphosphate aldolase (EC 4.1.2.13). Even a low dose of PCP (20 microM) decreased sharply activity of the mentioned key enzymes and intermediates' production in the cells of the studied strains with the use of both substrates. However, presence of clinoptilolites in the medium reduced significantly the biocide inhibition effect. Furthermore, in the medium with glucose, protection of intracellular metabolism with the help of sorbents was registered more clearly than with pectin. This can evidence for more mobile and simpler possibilities of accelerated production of necessary intermediates from glucose that are capable to induce activation of the key enzymatic reactions in cells utilizing selectively the substrates (which are different in accessibility and other characteristics) under the toxic agent effect.
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PMID:[Inhibition of carbohydrate metabilsm enzymes by pentachlorophenol in cells of pectinolytic bacteria from gastrointestinal tract of animals and effect of clinoptilolite adsorbents on this process]. 1729 Jul 94

Narrow substrate specificities often limit the use of enzymes in biocatalysis. To further the development of Escherichia coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase as a biocatalyst, the molecular determinants of substrate specificity were probed by mutagenesis. Our data demonstrate that S184 is located in the substrate-binding pocket and interacts with the phosphate moiety of KDPG, providing biochemical support for the binding model proposed on the basis of crystallographic data. An analysis of the substrate selectivity of the mutant enzymes indicates that alterations to the phosphate-binding site of KDPG aldolase changes the substrate selectivity. We report mutations that enhance catalysis of aldol cleavage of substrates lacking a phosphate moiety and demonstrate that electrophile reactivity correlates with the hydrophobicity of the substituted side chain. These mutations improve the selectivity for unnatural substrates as compared to KDPG by up to 2000-fold. Furthermore, the S184L KDPG aldolase mutant improves the catalytic efficiency for the synthesis of a precursor for nikkomycin by 40-fold, making it a useful biocatalyst for the preparation of fine chemicals.
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PMID:Mutagenesis of the phosphate-binding pocket of KDPG aldolase enhances selectivity for hydrophobic substrates. 1796

2-Keto-3-deoxy-6-phosphogluconate (KDPG) and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases catalyze an identical reaction differing in substrate specificity in only the configuration of a single stereocenter. However, the proteins show little sequence homology at the amino acid level. Here we investigate the determinants of substrate selectivity of these enzymes. The Escherichia coli KDPGal aldolase gene, cloned into a T7 expression vector and overexpressed in E. coli, catalyzes retro-aldol cleavage of the natural substrate, KDPGal, with values of k(cat)/K(M) and k(cat) of 1.9x10(4)M(-1)s(-1) and 4s(-1), respectively. In the synthetic direction, KDPGal aldolase efficiently catalyzes an aldol addition using a limited number of aldehyde substrates, including d-glyceraldehyde-3-phosphate (natural substrate), d-glyceraldehyde, glycolaldehyde, and 2-pyridinecarboxaldehyde. A preparative scale reaction between 2-pyridinecarboxaldehyde and pyruvate catalyzed by KDPGal aldolase produced the aldol adduct of the R stereochemistry in >99.7% ee, a result complementary to that observed using the related KDPG aldolase. The native crystal structure has been solved to a resolution of 2.4A and displays the same (alpha/beta)(8) topology, as KDPG aldolase. We have also determined a 2.1A structure of a Schiff base complex between the enzyme and its substrate. This model predicts that a single amino acid change, T161 in KDPG aldolase to V154 in KDPGal aldolase, plays an important role in determining the stereochemical course of enzyme catalysis and this prediction was borne out by site-directed mutagenesis studies. However, additional changes in the enzyme sequence are required to prepare an enzyme with both high catalytic efficiency and altered stereochemistry.
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PMID:Characterization and crystal structure of Escherichia coli KDPGal aldolase. 1798 70


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