In addition to the cleavage of 2-dehydro-3-deoxy-D-gluconate 6-phosphate, it is also found to naturally catalyze Schiff base formation between a lysine E-amino acid group and carbonyl compounds, decarboxylation of oxaloacetate, and exchange of solvent protons with the methyl hydrogen atoms of pyruvate.[2]
Nomenclature
The systematic name of this enzyme class is 2-dehydro-3-deoxy-D-gluconate-6-phosphate D-glyceraldehyde-3-phosphate-lyase (pyruvate-forming). Other names in common use include:
Active site structure with bound pyruvate (colors correspond to secondary structure, with cyan, magenta, and light pink referring to helices, sheets, and loops respectively)
KDPG Aldolase was recently determined to be a trimer through crystallographic three-fold symmetry, with 225 residues.[2][3] The enzyme was determined to have a molecular weight of 23,942.[4] The trimer is stabilized primarily through hydrophobic interactions. The molecule has tertiary folding similar to triosephosphate isomerase and the A-domain of pyruvate kinase, forming an eight strand α/β-barrel structure.[3][5] The α/β-barrel structure is capped on one side by the N-terminal helix. The other side, the carboxylic side, contains the active site.[6] Each subunit contains a phosphate-ion bound in position of the aldolase binding site.[7] It has been found that there are four cysteinyl groups present in each subunit, with two readily accessible and two buried in the subunit.[8]
The active site contains the zwitterionic pair Glu-45/Lys-133.[9] The Lysine, which is involved in the formation of the Schiff base is coordinated by a phosphate ion and two solvent water molecules.[6][7] The first water molecule serves as a shuttle between the Glutamate and the substrate, staying bound to the enzyme throughout the catalytic cycle.[7] The second water molecule is a product of the dehydration of the carbinolamine that leads to the formation of the Schiff base.[7] It also functions as the nucleophile during hydrolysis of the enzyme-product Schiff base, leading to the release of pyruvate.[7]
Active site showing interaction of pyruvate with zwitterionic pair Glu-45/Lys-133
One of the reactions KDPG Aldolase catalyzes, as in the Entner–Doudoroff pathway, is the reversible cleavage of 2-keto-3-deoxy-6-phosphogluconate (KDPG) into pyruvate and D-glyceraldehyde-3-phosphate.[9][10] This occurs through a stereospecific retro-aldol cleavage.[7] A proton transfer between the zwitterionic pair Glu-45/Lys-133 in the active site activates Lysine to serve as the nucleophile in the first step and Glutamate to aid in the base catalysis involved in the carbon-carbon cleavage.[9] Lysine Residue 133 serves as the nucleophile and attacks the carbonyl group of 2-Keto-3-deoxy-6-phosphogluconate to form a protonated carbinolamine intermediate, also known as a Schiff base intermediate.[7][9][10] The intermediate is stabilized by hydrogen bonding with residues in the active site.[9] A three carbon residue, glyceraldehyde 3-phosphate, is cleaved off through base catalysis with a water molecule and residue Glu-45.[7][9] The pyruvate is generated through the nucleophilic attack of water on the Schiff-base to reform a ketone. Aromatic interaction with Phe-135 ensures the stereospecific addition involved in the reverse process.[9]
KDPG aldolase has also been shown to catalyze the exchange of hydrogen atoms of the methyl groups of pyruvate with protons of the solvent.[10]
KDPG aldolase mechanism in the Entner–Doudoroff pathway
Evolutionary significance
History
Arguments have been made for both the convergent and divergent evolution of α/β-barrel structured enzymes such as KDPG Aldolase, triosephosphate isomerase, and the A-domain of pyruvate kinase.
Convergent evolution can lead to geometrically similar active sites while each enzyme has a distinct backbone conformation. Convergence to a common backbone structure, as is the case here however, has not been observed, although it is argues that it might be possible for a symmetrically repetitive structure as the one observed here.[11] The similarity in the folding of the three enzymes and the exceptional symmetry commonly suggests divergent evolution from a common ancestor. The functional similarity of the enzymes remains the strongest argument for divergent evolution.[11] All three enzymes activate a C–H bond adjacent to a carbonyl group. The active sites are located at the carboxylic ends of the β strands. Such congruence is in favor of divergent evolution.
Should the divergent evolution hypothesis prevail, this would suggest the existence of a class of enzymes with unrelated amino acid sequences yet analogous symmetrical structure and folding.[11]
Directed Evolution
KDPG aldolase has limited utility due to its high specificity for its natural substrates in the cleavage of KDPG and the reverse addition of D-glyceraldehyde-3-phosphate and pyruvate.[12] In vitro evolution has allowed KDPG aldolase to be converted into a more efficient aldolase with altered substrate specificity and stereoselectivity thereby improving its utility in asymmetric synthesis.[13] Rather than modifying the recognition site, the substrate is modified by moving the active site lysine from one β strand to a neighboring one.[13][14] The evolved aldolase is capable of accepting both D- and L-glyceraldehyde in their non-phosphorylated form.[15]
Related Research Articles
The Entner–Doudoroff pathway is a metabolic pathway that is most notable in Gram-negative bacteria, certain Gram-positive bacteria and archaea. Glucose is the substrate in the ED pathway and through a series of enzyme assisted chemical reactions it is catabolized into pyruvate. Entner and Doudoroff (1952) and MacGee and Doudoroff (1954) first reported the ED pathway in the bacterium Pseudomonas saccharophila. While originally thought to be just an alternative to glycolysis (EMP) and the pentose phosphate pathway (PPP), some studies now suggest that the original role of the EMP may have originally been about anabolism and repurposed over time to catabolism, meaning the ED pathway may be the older pathway. Recent studies have also shown the prevalence of the ED pathway may be more widespread than first predicted with evidence supporting the presence of the pathway in cyanobacteria, ferns, algae, mosses, and plants. Specifically, there is direct evidence that Hordeum vulgare uses the Entner–Doudoroff pathway.
Aldolase B also known as fructose-bisphosphate aldolase B or liver-type aldolase is one of three isoenzymes of the class I fructose 1,6-bisphosphate aldolase enzyme, and plays a key role in both glycolysis and gluconeogenesis. The generic fructose 1,6-bisphosphate aldolase enzyme catalyzes the reversible cleavage of fructose 1,6-bisphosphate (FBP) into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP) as well as the reversible cleavage of fructose 1-phosphate (F1P) into glyceraldehyde and dihydroxyacetone phosphate. In mammals, aldolase B is preferentially expressed in the liver, while aldolase A is expressed in muscle and erythrocytes and aldolase C is expressed in the brain. Slight differences in isozyme structure result in different activities for the two substrate molecules: FBP and fructose 1-phosphate. Aldolase B exhibits no preference and thus catalyzes both reactions, while aldolases A and C prefer FBP.
6-Phosphogluconate dehydrogenase (6PGD) is an enzyme in the pentose phosphate pathway. It forms ribulose 5-phosphate from 6-phosphogluconate:
Transaldolase is an enzyme of the non-oxidative phase of the pentose phosphate pathway. In humans, transaldolase is encoded by the TALDO1 gene.
In enzymology, a phosphogluconate dehydrogenase (decarboxylating) (EC 1.1.1.44) is an enzyme that catalyzes the chemical reaction
In enzymology, a 2-dehydro-3-deoxy-D-gluconate 5-dehydrogenase (EC 1.1.1.127) is an enzyme that catalyzes the chemical reaction
In enzymology, a 2-dehydro-3-deoxy-D-gluconate 6-dehydrogenase (EC 1.1.1.126) is an enzyme that catalyzes the chemical reaction
The enzyme Glucosaminate ammonia-lyase (EC 4.3.1.9) catalyzes the chemical reaction
The enzyme 2-dehydro-3-deoxy-6-phosphogalactonate aldolase catalyzes the chemical reaction
The enzyme 2-dehydro-3-deoxy-D-pentonate aldolase catalyzes the chemical reaction
The enzyme 2-dehydro-3-deoxyglucarate aldolase catalyzes the chemical reaction
The enzyme 2-dehydro-3-deoxy-L-pentonate aldolase catalyzes the chemical reaction
The enzyme 5-dehydro-2-deoxyphosphogluconate aldolase catalyzes the chemical reaction
The enzyme deoxyribose-phosphate aldolase catalyzes the reversible chemical reaction
The enzyme phosphogluconate dehydratase (EC 4.2.1.12) catalyzes the chemical reaction
In enzymology, a 1-deoxy-d-xylulose-5-phosphate synthase (EC 2.2.1.7) is an enzyme in the non-mevalonate pathway that catalyzes the chemical reaction
In enzymology, a 3-deoxy-8-phosphooctulonate synthase (EC 2.5.1.55) is an enzyme that catalyzes the chemical reaction
In enzymology, a 2-dehydro-3-deoxygluconokinase is an enzyme that catalyzes the chemical reaction
Aldolase C, fructose-bisphosphate, is an enzyme that, in humans, is encoded by the ALDOC gene on chromosome 17. This gene encodes a member of the class I fructose-bisphosphate aldolase gene family. Expressed specifically in the hippocampus and Purkinje cells of the brain, the encoded protein is a glycolytic enzyme that catalyzes the reversible aldol cleavage of fructose 1,6-bisphosphate and fructose-1-phosphate to dihydroxyacetone phosphate and either glyceraldehyde 3-phosphate or glyceraldehyde, respectively.[provided by RefSeq, Jul 2008]
3-Deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase is the first enzyme in a series of metabolic reactions known as the shikimate pathway, which is responsible for the biosynthesis of the amino acids phenylalanine, tyrosine, and tryptophan. Since it is the first enzyme in the shikimate pathway, it controls the amount of carbon entering the pathway. Enzyme inhibition is the primary method of regulating the amount of carbon entering the pathway. Forms of this enzyme differ between organisms, but can be considered DAHP synthase based upon the reaction that is catalyzed by this enzyme.
1 2 Mavridis IM, Tulinsky A (October 1976). "The folding and quaternary structure of trimeric 2-keto-3-deoxy-6-phosphogluconic aldolase at 3.5-A resolution". Biochemistry. 15 (20): 4410–7. doi:10.1021/bi00665a010. PMID974067.
1 2 Mavridis IM, Hatada MH, Tulinsky A, Lebioda L (December 1982). "Structure of 2-keto-3-deoxy-6-phosphogluconate aldolase at 2 . 8 A resolution". J. Mol. Biol. 162 (2): 419–44. doi:10.1016/0022-2836(82)90536-8. PMID7161801.
↑ Möhler H, Decker K, Wood WA (July 1972). "Structure of 2-keto-3-deoxy-6-phosphogluconate aldolase. IV. Structural features revealed by treatment with urea and Ellman's reagent". Arch. Biochem. Biophys. 151 (1): 251–60. doi:10.1016/0003-9861(72)90495-x. PMID5044518.
1 2 3 Lebioda L, Hatada MH, Tulinsky A, Mavridis IM (December 1982). "Comparison of the folding of 2-keto-3-deoxy-6-phosphogluconate aldolase, triosephosphate isomerase and pyruvate kinase. Implications in molecular evolution". J. Mol. Biol. 162 (2): 445–58. doi:10.1016/0022-2836(82)90537-X. PMID7161802.
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