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PubMed2003

Structure-based mutagenesis approaches toward expanding the substrate specificity of D-2-deoxyribose-5-phosphate aldolase.

2-Deoxyribose-5-phosphate aldolase (DERA, EC 4.1.2.4) catalyzes the reversible aldol reaction between acetaldehyde and D-glyceraldehyde-3-phosphate to generate D-2-deoxyribose-5-phosphate. It is unique among the aldolases as it catalyzes the reversible asymmetric aldol addition reaction of two aldehydes. In order to expand the substrate scope and stereoselectivity of DERA, structure-based substrate design as well as site-specific mutation has been investigated. Using the 1.05 A crystal structure of DERA in complex with its natural substrate as a guide, five site-directed mutants were designed in order to improve its activity with the unnatural nonphosphorylated substrate, D-2-deoxyribose. Of these, the S238D variant exhibited a 2.5-fold improvement over the wild-type enzyme in the retroaldol reaction of 2-deoxyribose. Interestingly, this S238D mutant enzyme was shown to accept 3-azidopropinaldehyde as a substrate in a sequential asymmetric aldol reaction to form a deoxy-azidoethyl pyranose, which is a precursor to the corresponding lactone and the cholesterol-lowering agent Lipitor. This azidoaldehyde is not a substrate for the wild-type enzyme. Another structure-based design of new nonphosphorylated substrates was focused on the aldol reaction with inversion in enantioselectivity using the wild type or the S238D variant as the catalyst and 2-methyl-substituted aldehydes as substrates. An example was demonstrated in the asymmetric synthesis of a deoxypyranose as a new effective synthon for the total synthesis of epothilones. In addition, to facilitate the discovery of new enzymatic reactions, the engineered E. coli strain SELECT (Deltaace, adhC, DE3) was developed to be used in the future for selection of DERA variants with novel nonphosphorylated acceptor specificity.

Aldehyde-Lyases↗
PubMed1976

The folding and quaternary structure of trimeric 2-keto-3-deoxy-6-phosphogluconic aldolase at 3.5-A resolution.

An X-ray crystallographic structure determination has been carried out on 2-keto-3-deoxy-6-phosphogluconic (KDPG) aldolase at 3.5-A resolution using the multiple isomorphous replacement method with three heavy atom derivatives along with anomalous dispersion contributions from two of the derivatives. Crystals grown from ammonium sulfate-phosphate buffered (pH 3.5) solutions were: cubic, a= 103.40 (4) A, space group P213. KDPG aldolase consists of trimeric heterologous assemblages utilizing crystallographic threefold symmetry. The overall profile of the oligomeric structure viewed down the threefold axis resembles that of a ship propeller while the subunits are approximate irregular oblate ellipsoids (25 X 45 X 45 A). The folding of most of the polypeptide chain was traced unambiguously. Secondary structural features consist of nine helical regions (75 residues, 35%) and a pair of two parallel chains. The subunit contains a long empty channel which is about 9 X 9 X 30 A with one of the pair of parallel chains forming part of the wall. Three mercury binding sites are located in this channel. These might correspond to the two readily accessible and one of the two buried cysteine residues of each subunit. The channel terminates with another cavity of about 8 X 10 X 25 A near the surface of the oligomeric structure. The regions of the subunits near the threefold axis are characterized by a high degree of secondary structural organization and these make close intersubunit contacts. Quarternary interactions are due mainly to side-chain interactions of helices.

Aldehyde-Lyases↗
PubMed1975

Bromopyruvate inactivation of 2-keto-3-deoxy-6-phosphogalactonate aldolase of Pseudomonas saccharophila. Kinetics and stereochemistry.

The enzyme 2-keto-3-deoxy-6-phosphogalactonate aldolase of Pseudomonas saccharophila is inactivated by the substrate analog beta-bromopyruvate, which satisfies several criteria of being an active site directed reagent. The inactivation exhibits saturation kinetics, and both bromopyruvate and pyruvate (substrate) compete for free enzyme. Upon prolonged incubation, inactivation is virtually complete. The Kinact for bromopyruvate is 12 mM and the minimum inactivation half-time is 16 min with a k of 0.0433 min minus 1. Bromopyruvate is also a substrate for the enzyme in that 3(R,S)-[3-3H2]bromopyruvate is asymmetrically detritiated by the enzyme yielding 3(S)-[3-3H,H]bromopyruvate concomitant with inactivation. At various concentrations of bromopyruvate which affect the inactivation rate, the ratio of nanomoles of bromopyruvate turned over/unit of enzyme inactivated remains constant averaging 12:1, consistent with both inactivation and catalysis occurring at a single protein site, the catalytic site. The above value does not take into account a possible hydrogen isotope effect and is not thus an absolute value. The stereochemistry of bromopyruvate turnover catalyzed by this enzyme is the same as that for 2-keto-3-deoxy-6-phosphogluconate aldolase of P. putida. This fact provides the first evidence that the pyruvate-specific portions of the two active sites may have evolved from a common precursor.

Aldehyde-Lyases↗
PubMed2001

The structure of L-ribulose-5-phosphate 4-epimerase: an aldolase-like platform for epimerization.

The structure of L-ribulose-5-phosphate 4-epimerase from E. coli has been solved to 2.4 A resolution using X-ray diffraction data. The structure is homo-tetrameric and displays C(4) symmetry. Each subunit has a single domain comprised of a central beta-sheet flanked on either side by layers of alpha-helices. The active site is identified by the position of the catalytic zinc residue and is located at the interface between two adjacent subunits. A remarkable feature of the structure is that it shows a very close resemblance to that of L-fuculose-1-phosphate aldolase. This is consistent with the notion that both enzymes belong to a superfamily of epimerases/aldolases that catalyze carbon-carbon bond cleavage reactions via a metal-stabilized enolate intermediate. Detailed inspection of the epimerase structure, however, indicates that despite the close overall structural similarity to class II aldolases, the enzyme has evolved distinct active site features that promote its particular chemistry.

Aldehyde-Lyases↗
PubMed1998

Crystal structure and reaction mechanism of 7,8-dihydroneopterin aldolase from Staphylococcus aureus.

Dihydroneopterin aldolase catalyzes the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin during the de novo synthesis of folic acid from guanosine triphosphate. The gene encoding the dihydroneopterin aldolase from S. aureus has been cloned, sequenced and expressed in Escherichia coli. The protein has been purified for biochemical characterization and its X-ray structure determined at 1.65 A resolution. The protein forms an octamer of 110,000 Mr molecular weight. Four molecules assemble into a ring, and two rings come together to give a cylinder with a hole of at least 13 A diameter. The structure of the binary complex with the product 6-hydroxymethyl-7,8-dihydropterin has defined the location of the active site. The structural information and results of site directed mutagenesis allow an enzyme reaction mechanism to be proposed.

Aldehyde-Lyases↗
PubMed1998

Gene cloning, biochemical characterization and physiological role of a thermostable low-specificity L-threonine aldolase from Escherichia coli.

The ltaE gene encoding for a thermostable low-specificity L-threonine aldolase, which catalyzes the cleavage of L-threonine/L-allo-threonine to glycine and acetaldehyde, was cloned from Escherichia coli GS245 by the polymerase chain reaction. Construction and expression of the plasmid pLTAE, which contained the ltaE gene under the control of the lac promoter, resulted in a 227-fold increase in the specific activity above the level detected in E. coli cells containing the control vector. The enzyme is thermostable: it retained its full activity upon heating at 60 degrees C for 1 h. The enzyme was thus feasibly purified to homogeneity by heat treatment and butyl-Toyopearl column chromatography, and characterized. To reveal the physiological role of the enzyme, gene disruption was performed. Knockout of the ltaE gene of wild-type E. coli did not effect the cellular growth rate, while disruption of the ltaE gene of E. coli GS245, whose serine hydroxymethyltransferase gene was knocked out, caused a significant decrease in the cellular growth rate, suggesting that the threonine aldolase is not a major source of cellular glycine in wild-type E. coli but catalyzes an alternative pathway for cellular glycine when serine hydroxymethyltransferase is inert.

Bacterial Proteins↗
PubMed2003

Crystal structure of a bifunctional aldolase-dehydrogenase: sequestering a reactive and volatile intermediate.

The crystal structure of the bifunctional enzyme 4-hydroxy-2-ketovalerate aldolase (DmpG)/acylating acetaldehyde dehydrogenase (DmpF), which is involved in the bacterial degradation of toxic aromatic compounds, has been determined by multiwavelength anomalous dispersion (MAD) techniques and refined to 1.7-A resolution. Structures of the two polypeptides represent a previously unrecognized subclass of metal-dependent aldolases, and of a CoA-dependent dehydrogenase. The structure reveals a mixed state of NAD+ binding to the DmpF protomer. Domain movements associated with cofactor binding in the DmpF protomer may be correlated with channeling and activity at the DmpG protomer. In the presence of NAD+ a 29-A-long sequestered tunnel links the two active sites. Two barriers are visible along the tunnel and suggest control points for the movement of the reactive and volatile acetaldehyde intermediate between the two active sites.

Acetaldehyde↗
PubMed1990

Activation of Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase by inorganic phosphate.

Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17], one of the metal ion-requiring aldolases, is markedly activated by Pi. The activation is reversible and can be observed in every step of enzyme purification. The extent of activation is almost independent of the metal ion used, but varies with each substrate. The cleavage of l-4-carboxy-4-hydroxy-2-oxoadipate, a physiological substrate of the enzyme, is most strongly activated: Pi gives a hyperbolic activation curve with an activation constant of 0.36 mM and a maximum activation of about 65-fold. Arsenate, phosphorous acid, bicarbonate, acetyl phosphate, thiamine diphosphate, ADP, PPi, and ATP are also effective to various extents. These anions appear to be effective in the free form but not in the metal ion-complex. Many organic and inorganic anions are ineffective. Pi causes parallel increases in Vmax and in Km for substrate or metal ion with a concomitant shift of the optimum pH toward the alkaline side, and the enhancement of activity is closely correlated with the shift of optimum pH. Pi induces no gross change of molecular form of the enzyme protein as evaluated from gel filtration, PAGE, UV, fluorescence, and CD spectral data. Based on these findings, the mechanism and the physiological meaning of the observed activation are discussed.

Chromatography, Gel↗
PubMed1991

Chemical modification of Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase by diethyl pyrocarbonate.

Diethyl pyrocarbonate inactivates Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17] by a simple bimolecular reaction. The inactivation is not reversed by hydroxylamine. The pH curve of inactivation indicates the involvement of a residue with a pK of 8.8. Several lines of evidence show that the inactivation is due to the modification of epsilon-amino groups of lysyl residues. Although histidyl residue is also modified, this is not directly correlated to the inactivation. No cysteinyl, tyrosyl, or tryptophyl residue or alpha-amino group is significantly modified. The modification of three lysyl residues per enzyme subunit results in the complete loss of aldolase activity toward various 4-hydroxy-2-oxo acid substrates, whereas oxaloacetate beta-decarboxylase activity associated with the enzyme is not inhibited by this modification. Statistical analysis suggests that only one of the three lysyl residues is essential for activity. l-4-Carboxy-4-hydroxy-2-oxoadipate, a physiological substrate for the enzyme, strongly protects the enzyme against inactivation. Pi as an activator of the enzyme shows no specific protection. The molecular weight of the enzyme, Km for substrate or Mg2+, and activation constant for Pi are virtually unaltered after modification. These results suggest that the modification occurs at or near the active site and that the essential lysyl residue is involved in interaction with the hydroxyl group but not with the oxal group of the substrate.

Amino Acids↗
PubMed2003

The sugar-metabolic enzymes aldolase and triose-phosphate isomerase are targets of glutathionylation in Arabidopsis thaliana: detection using biotinylated glutathione.

GSH has multiple actions in physiological responses of plants, but the molecular mechanisms are not fully understood. GSH plays an important role in functional alteration of proteins by reversible covalent incorporation (glutathionylation) in vertebrate cells. To investigate the function of glutathionylation in plant cells, we examined glutathionylated proteins in the suspension-cultured cells of Arabidopsis using biotinylated GSH. Biotinylated GSH was incorporated into about 20 proteins. Two of these proteins were identified as the key enzymes for sugar metabolism, triose-phosphate isomerase (TPI) and putative plastidic aldolase. Recombinant TPI was inactivated by GSSG, and it was reactivated by GSH. The physiological roles of glutathionylation of TPI and aldolase in sugar metabolism are discussed.

Aldehyde-Lyases↗
PubMed2004

Folate biosynthesis in higher plants. cDNA cloning, heterologous expression, and characterization of dihydroneopterin aldolases.

Dihydroneopterin aldolase (EC 4.1.2.25) is one of the enzymes of folate synthesis that remains to be cloned and characterized from plants. This enzyme catalyzes conversion of 7,8-dihydroneopterin (DHN) to 6-hydroxymethyl-7,8-dihydropterin, and is encoded by the folB gene in Escherichia coli. The E. coli FolB protein also mediates epimerization of DHN to 7,8-dihydromonapterin. Searches of the Arabidopsis genome detected three genes encoding substantially diverged FolB homologs (AtFolB1-3, sharing 57%-73% identity), for which cDNAs were isolated. A fourth cDNA specifying a FolB-like protein (LeFolB1) was obtained from tomato (Lycopersicon esculentum) by reverse transcription-PCR. When overproduced in E. coli, recombinant AtFolB1, AtFolB2, and LeFolB1 proteins all had both dihydroneopterin aldolase and epimerase activities, and carried out the aldol cleavage reaction on the epimerization product, 7,8-dihydromonapterin, as well as on DHN. AtFolB3, however, could not be expressed in active form. Size exclusion chromatography indicated that the plant enzyme is an octamer, like the bacterial enzyme. Quantifying expression of the Arabidopsis genes by real-time reverse transcription-PCR showed that AtFolB1 and AtFolB2 messages occur at low levels throughout the plant, whereas the AtFolB3 mRNA was detected only in siliques and only with an extremely low abundance. Sequence comparisons and phylogenetic analysis of FolB homologs from 16 plants indicated that their N-terminal regions are highly variable, and that most species have a small number of FolB genes that diverged after separation of the lineages leading to families. The substantial divergence of FolB homologs in Arabidopsis and other plants suggests that some of them may act on substrates other than DHN.

Aldehyde-Lyases↗
PubMed1972

Chloroplast and Cytoplasmic Enzymes: IV. Pea Leaf Fructose 1,6-Diphosphate Aldolases.

Several peaks of aldolase activity are found in the isoelectric focusing pattern of pea (Pisum sativum) leaf chloroplast extracts. One peak, separated by 0.5 pH unit from the major chloroplast aldolase peak, is found when cytoplasmic extracts are focused. The chloroplast and cytoplasmic enzymes have a pH 7.4 optimum with fructose 1,6-diphosphate. The Michaelis constant for fructose-1,6-diphosphate is 19 muM for the chloroplast, 21 muM for the cytoplasmic enzyme, and for sedoheptulose 1,7-diphosphate, 8 muM for the chloroplast enzyme, 18 muM for the cytoplasmic enzyme. Both enzymes are inhibited by d-glyceraldehyde 3-phosphate and by ribulose 1,5-diphosphate. The similarity in the catalytic properties of the isoenzymes suggests that both enzymes have an amphibolic role in carbon metabolism in the green leaf.

Journal Article↗
PubMed2001

Crystallization and preliminary X-ray analysis of dmpFG-encoded 4-hydroxy-2-ketovalerate aldolase--aldehyde dehydrogenase (acylating) from Pseudomonas sp. strain CF600.

The final two steps of the meta-cleavage pathway for catechol degradation in Pseudomonas sp. strain CF600 involve the conversion of 4-hydroxy-2-ketovalerate to pyruvate and acetyl coenzyme A by the enzymes 4-hydroxy-2-ketovalerate aldolase and NAD(+)-dependent acylating aldehyde dehydrogenase. Biochemical studies indicate that these two enzymes comprise a bifunctional heterodimer (DmpFG, molecular mass 71 kDa) and suggest that the product of the aldolase reaction is transferred to the dehydrogenase active site via a channeling mechanism. Crystals of the DmpFG complex grow in multiple fan-like clusters of thin plates by the hanging-drop method and are improved by streak-seeding. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 102.0, b = 140.7, c = 191.3 A, and diffract to 2.1 A resolution. The asymmetric unit contains four DmpFG heterodimers. Heavy-atom derivative screening identified three isomorphous derivatives.

Aldehyde Oxidoreductases↗
PubMed2003

Structure of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from Pseudomonas putida.

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.

Aldehyde-Lyases↗
PubMed1999

Rhombohedral crystals of 2-dehydro-3-deoxygalactarate aldolase from Escherichia coli.

2-Dehydro-3-deoxygalactarate (DDG) aldolase (E.C. 4.1.2.20) catalyzes the reversible aldol cleavage of DDG and 2-dehydro-3-deoxyglucarate to pyruvate and tartronic semialdehyde. Rhombohedral crystals of recombinant DDG aldolase from Escherichia coli K-12 were obtained. The crystals belong to space group R32 with unit-cell parameters a = 93 A, alpha = 85 degrees. The crystals diffract to beyond 1.8 A resolution on a Cu Kalpha rotating-anode generator. The asymmetric unit is likely to contain two molecules, corresponding to a packing density of 1.34 A3 Da-1.

Aldehyde-Lyases↗
PubMed1997

Purification and characterization of L-allo-threonine aldolase from Aeromonas jandaei DK-39.

L-allo-Threonine aldolase (L-allo-threonine acetaldehyde-lyase), which exhibited specificity for L-allo-threonine but not for L-threonine, was purified from a cell-free extract of Aeromonas jandaei DK-39. The purified enzyme catalyzed the aldol cleavage reaction of L-allo-threonine (K(m) = 1.45 mM, Vmax = 45.2 mumol min-1 mg-1). The activity of the enzyme was inhibited by carbonyl reagents, which suggests that pyridoxal-5'-phosphate participates in the enzymatic reaction. The enzyme does not act on either L-serine or L-threonine, and thus it can be distinguished from serine hydroxy-methyltransferase (L-serine:tetrahydrofolate 5,10-hydroxy-methyltransferase, EC 2.1.2.1) or L-threonine aldolase (EC 4.1.2.5).

Aeromonas↗
PubMed1998

Gene cloning, nucleotide sequencing, and purification and characterization of the low-specificity L-threonine aldolase from Pseudomonas sp. strain NCIMB 10558.

A low-specificity L-threonine aldolase (L-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia coli cells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5'-phosphate as a coenzyme, is strictly L specific at the alpha position, whereas it cannot distinguish between threo and erythro forms at the beta position. In addition to threonine, the enzyme also acts on various other L-beta-hydroxy-alpha-amino acids, including L-beta-3,4-dihydroxyphenylserine, L-beta-3,4-methylenedioxyphenylserine, and L-beta-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificity L-TA from Saccharomyces cerevisiae, L-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity L-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of the L-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5'-phosphate of the enzyme to catalyze the reversible aldol reaction.

Amino Acid Sequence↗