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PubMed2004

Development of an efficient, scalable, aldolase-catalyzed process for enantioselective synthesis of statin intermediates.

A process is reported for efficient, enantioselective production of key intermediates for the common chiral side chain of statin-type cholesterol-lowering drugs such as Lipitor (atorvastatin) and Crestor (rosuvastatin). The process features a one-pot tandem aldol reaction catalyzed by a deoxyribose-5-phosphate aldolase (DERA) to form a 6-carbon intermediate with installation of two stereogenic centers from 2-carbon starting materials. An improvement of almost 400-fold in volumetric productivity relative to the published enzymatic reaction conditions has been achieved, resulting in a commercially attractive process that has been run on up to a 100-g scale in a single batch at a rate of 30.6 g/liter per h. Catalyst load has been improved by 10-fold as well, from 20 to 2.0 wt % DERA. These improvements were achieved by a combination of discovery from environmental DNA of DERAs with improved activity and reaction optimization to overcome substrate inhibition. The two stereogenic centers are set by DERA with enantiomeric excess at >99.9% and diastereomeric excess at 96.6%. In addition, down-stream chemical steps have been developed to convert the enzymatic product efficiently to versatile intermediates applicable to preparation of atorvastatin and rosuvastatin.

Amino Acid Sequence↗
PubMed1979

Paracatalytic modification of aldolase: a side reaction of the catalytic cycle resulting in irreversible blocking of two active-site lysyl residues.

Paracatalytic enzyme modifications result from the oxidation of enzyme-substrate carbanions by extrinsic oxidants. During the oxidation of enzyme-activated substrates, transiently reactive intermediates are generated which, without being released from the enzyme, modify groups at the active site. For enzymes producing carbanion intermediates, the combination of the normal substrate with a suitable electron acceptor has thus been proposed as a highly specific binary system for their active site-directed modification. In this study, the structural features of paracatalytically modified fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) from rabbit muscle have been elucidated. This enzyme is completely inactivated within 60 min in the presence of fructose 1,6-bisphosphate in saturating concentration and 0.5 mM hexacyanoferrate(III) (pH 7.6, 25 degrees C). The inactivation is caused by covalent incorporation of one triosephosphate derivative per subunit. Peptide analysis showed that the triosephosphate derivative forms an intrachain crosslink between lysine-146 and lysine-227. According to previous independent experimental evidence, both lysyl residues are located at the active site: the epsilon-amino group of lysine-227 forms a Schiff base intermediate with the carbonyl group of the substrate [Lai, C. Y., Nakai, N. & Chang, D. (1974) Science 183, 1204-1206] and alkylation of lysine-146 by the affinity labeling reagent N-bromoacetylethanolamine phosphate inactivates the enzyme [Hartman, F. C. & Brown, J. P. (1976) J. Biol. Chem. 251, 3057-3062]. The present data thus establish paracatalytic modification as a mode of active site-directed enzyme modification.

Animals↗
PubMed1996

An intronic enhancer essential for tissue-specific expression of the aldolase B transgenes.

Expression in mice of transgenes directed by regulatory regions of the rat aldolase B gene requires the presence of a B element located in the first intron, while constructs devoid of this intronic enhancer are silent. Histo- and immunochemical staining of transgenic tissue sections showed that the longer transgene was expressed in the proximal tubular cells of the kidney, enterocytes located in small intestine villi and liver parenchymal cells. In the liver, a maximal expression was observed in perivenous hepatocytes, while the transgene was weakly active in periportal hepatocytes, which reproduced the pattern of functional zonation already reported for other glycolytic and gluconeogenic genes in the liver. We also established that the transgene retained the necessary elements for a correct chronological expression during development but was lacking elements necessary for activation by high carbohydrate diet. Instead, transgene expression was paradoxically stimulated in fasted animals, suggesting that the endogenous gene, which must be active under both glycolytic and gluconeogenic conditions, could possess distinct elements activating it in fasted as well as in carbohydrate-fed animals; the former element might be conserved in the transgene and the latter one might be lost.

Aging↗
PubMed1997

Carbon isotope effects on the fructose-1,6-bisphosphate aldolase reaction, origin for non-statistical 13C distributions in carbohydrates.

The kinetic and equilibrium isotope effects on the fructose-1, 6-bisphosphate aldolase reaction have been determined using the rabbit muscle enzyme. The natural 13C abundance for both atoms participating in the bond splitting were measured in position C-1 of dihydroxyacetone phosphate and glyceraldehyde 3-P after irreversible conversion to glycerol-3-P and 3-phosphoglycerate, respectively, and chemical degradation. The carbon isotope effects were determined comparing the 13C content of the corresponding positions after partial and complete turnover, and after complete equilibration of the reactants. 13(Vmax/Km) on C-3 was 1.016 +/- 0.007 and 0.997 +/- 0.009 on position C-4, and the equilibrium isotope effects K12/K13 on these positions were 1.0036 +/- 0.0002 and 1.0049 +/- 0.0001. The observed kinetic isotope effect on C-3 is discussed to originate from the formation of the enamine, which comes to equilibrium before the rate determining release of glyceraldehyde 3-P from the ternary complex. The equilibrium isotope effect is seen as the reason for an earlier-found relative 13C enrichment in position C-3 and C-4 of glucose and for varying enrichments in 13C of carbohydrates from different compartments of cells. The kinetic isotope effect is suggested to cause 13C discriminations in the C-3 pool in context with the hexose formation in competition with other dihydroxyacetone phosphate turnover reactions.

Animals↗
PubMed2002

Cloning and prokaryotic expression of a salt-induced cDNA encoding a chloroplastic fructose-1,6-diphosphate aldolase in Dunaliella salina (Chlorophyta).

A salt-induced fructose-1,6-diphosphate (FDP) aldolase cDNA (DsALDP) in Dunaliella salina was cloned by suppression subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE) techniques. Sequence analysis of DsALDP revealed that the 1520 bp cDNA had an open reading frame (ORF) of 327 amino acid residues. BLAST Search showed that DsALDP shared an amino acid identity (73-66%) with AldP in other plants. Alignment with homologues in other plants indicated that all the conserved substrate-specific binding sites could also be found in DsALDP. Phylogenetic analysis further confirmed the deduced amino acid sequence of the D. salina DsALDP gene belonged to the same subfamily to AldP of other green algae. Southern blot analysis suggested possible presence of the D. salina DsALDP gene as a few copies and Northern blot analysis confirmed salt-induced expression pattern at the transcriptional level. A 62 kDa fusion protein generated by adding a Trx-His.tag at the N-terminal of DsALDP was induced by IPTG in Escherichia coli BL21. An improvement of salt tolerance in E. coli expressing DsALDP fusion protein was observed.

Amino Acid Sequence↗
PubMed1989

Pediatric reference ranges for serum aldolase.

Most investigators have found that serum aldolase levels normally are higher in children than in adults. Although several earlier studies have included sufficient subjects to establish statistically reliable reference ranges, pediatric reference ranges have not been made available for the now widely used assay method of Pinto and associates (Clin Chem 1969;15:349-360.). The authors present age-related ranges determined by this method.

Adolescent↗
PubMed1994

Initiation zone of DNA replication at the aldolase B locus encompasses transcription promoter region.

Aldolase A (AldB) gene is one of the liver-specific genes, which is activated in the fetal stage. As a first step to investigate the functional relationship between transcription and DNA replication, we intended to determine the initiation zone of replication nearest to the AldB gene region. BrdU-labeled nascent DNA was obtained from G1/S arrested hepatoma cells at various times after entering S phase. Hybridization of the newly synthesized, BrdU-labeled DNA with probes corresponding to regions spanning about 26 Kb, revealed that replication zone locates within the AldB gene region. This result, together with the result of hybridization of nascent DNA obtained by alkaline sucrose density-gradient centrifugation, suggested that the initiation zone is located within a more defined region (about 1.0 Kb) containing AldB promoter. In the predicted initiation zone, a purine-rich element which shows high homology to known mammalian origin sequences and other replication components are found. Further, autonomously replicating activity of this initiation zone was examined by DNA transfection. The results showed that the predicted initiation zone confers replication initiation in Cos-1 cells.

Animals↗
PubMed1994

Activity of the rat liver-specific aldolase B promoter is restrained by HNF3.

Although it contains binding sites for HNF1, NFY and C/EBP/DBP, the proximal promoter of the aldolase B gene is surprisingly weak when tested by transient transfection in differentiated hepatoma cells. This low activity could be due to overlapping between HNF1 and HNF3 binding sites in element PAB, from -127 to -103 bp with respect to the cap site. Replacement of the PAB region by a consensus HNF1 binding site unable to bind HNF3, results in a 30 fold activation of the promoter, in accordance with the hypothesis that activity of the wild-type promoter is normally restrained by HNF3 binding to PAB competitively with HNF1. Consistently, transactivation of the wild-type promoter by excess HNF1 is very high, most likely due to the displacement of HNF3, while the construct with the exclusive HNF1 binding site is weakly transactivated by HNF1. The inhibitory effect of HNF3 on HNF1-dependent transactivation is clearly due to competition between these two factors for binding to mutually exclusive, overlapping sites; indeed, when HNF1 and HNF3 sites are contiguous and not overlapping, the resulting promoter is as active as the one containing an exclusive HNF1 binding site. A construct in which PAB has been replaced by an exclusive HNF3 binding site is weakly expressed and is insensitive to HNF3 hyperexpression. DBP-dependent transactivation, finally, is independent of the nature of the element present in the PAB region.

Animals↗
PubMed2002

Functional domains involved in the interaction between Orc1 and transcriptional repressor AlF-C that bind to an origin/promoter of the rat aldolase B gene.

The promoter of the rat aldolase B (AldB) gene functions in vivo as an origin of DNA replication in the cells in which transcription of the gene is repressed. Previously, we identified two closely related DNA-binding proteins, AlF-C1 and AlF-C2, which repressed the AldB gene promoter. We also reported that the binding site of these proteins, site C, is one of the required DNA elements of the AldB gene origin/promoter for autonomously replicating activity in transfected cells. In the present study, we show that AlF-C1 and AlF-C2 bind directly to Orc1, a subunit of the origin recognition complex (ORC). Deletion analyses revealed a functional domain in AlF-C2 for binding to Orc1, which is located separately from the DNA-binding domain. In addition, we found a novel protein-interacting domain in Orc1 required for the binding of AlF-C2, which was conserved in human, mouse and Chinese hamster, but not in Drosophila, frog and yeast. Thus, it is assumed that in mammalian cells, sequence- specific DNA-binding proteins are involved in recruiting ORC to regulate replication initiation and/or transcription repression.

Amino Acid Sequence↗
PubMed1999

Fructose 1,6-bisphosphate aldolase is a heparin-binding protein.

Proteins with affinity to heparin under physiological conditions were isolated from bovine cerebral cortex. First, the extract of cerebral cortex was applied to a chondroitin polysulfate column under physiological conditions. Then, the pass-through fraction was applied to a heparin column. Among the bands on SDS polyacrylamide gel electrophoresis of the fraction bound to the heparin column, the major one was identified as fructose 1,6-bisphosphate aldolase (FPA), a cytosolic enzyme involved in the glycolytic pathway. The results indicated that FPA is a heparin-binding protein which exhibits no affinity to chondroitin polysulfate. The results of affinity chromatographies revealed that FPA binds to intact heparin and modified heparins desulfated at C2 OH of the iduronic acid residue or at C6 OH or C2 NH2 of the glucosamine residue. When 6-O-desulfated heparin was employed as the affinity ligand, a single peak having FPA activity was isolated from the extract of bovine cerebral cortex. By further Mono Q chromatography and Superdex gel-filtration, five isoenzymes were purified with more than 50% recovery. These isoenzymes were identified as FPA A4, A3C1, A2C2, A1C3, and C4 by native electrophoresis with and without 4 M urea and subsequent amino acid sequence analysis. The use of 6-O-desulfated heparin affinity chromatography thus facilitated the purification of FPA.

Animals↗
PubMed1990

Purification and properties of 4-hydroxy-4-methyl-2-oxoglutarate aldolase from Pseudomonas ochraceae grown on phthalate.

4-Hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17] has been purified to homogeneity (about 770-fold purification, yield 11.4%) from Pseudomonas ochraceae grown on phthalate. The enzyme has a molecular weight of 160,000 (gel filtration on Bio-Gel A-1.5m), a subunit molecular weight of 26,000 (SDS-PAGE) and an isoelectric point of 5.0 (isoelectric focusing). The enzyme requires divalent metal ions such as Mg2+, Mn2+, Co2+, Zn2+, and Cd2+ for activity. The enzyme actively cleaves 4-carboxy-4-hydroxy-2-oxoadipate, a physiological substrate of the enzyme, to give pyruvate and oxaloacetate, but shows much lower affinity for 4-hydroxy-4-methyl-2-oxoglutarate. 4-Hydroxy-2-oxoglutarate is cleaved at a low rate to pyruvate and glyoxylate. The l-isomers of the substrates are preferentially cleaved rather than the d-isomers as determined polarimetrically. The enzyme reactions are reversible: the equilibrium constants (pH 8.0, 25 C) for the HMG and HG cleavage reactions are about 0.07 and 0.03 M, respectively, whereas no equilibrium is observed with CHA due to oxaloacetate beta-decarboxylase activity associated with the enzyme. The enzyme activity is hardly affected by thiols and thiol reagents. The non-enzymatic cleavage reaction caused by various metal ions has also been studied to examine the mechanistic similarity to the enzymatic reaction.

Chemical Phenomena↗
PubMed1995

Drosophila melanogaster aldolase: characterization of the isozymes alpha, beta, and gamma generated from a single gene.

Three isozymic forms, alpha, beta, and gamma, of Drosophila melanogaster aldolase are produced from a single gene by alternative usage of the triple exons 4 (4 alpha, 4 beta, and 4 gamma) [Shaw-Lee et al. (1992) J. Biol. Chem. 267, 3959-3967; Kim et al. (1992) Mol. Cell. Biol. 12, 773-783; Kai et al. (1992) J. Biochem. 112,677-688]. The expression plasmids for the respective isozymes were transfected into Escherichia coli cells, and the isozymes alpha and beta were purified to homogeneity by a simple procedure, though isozyme gamma was only partially purified. These isozymes are active towards two substrates, fructose-1,6-bisphosphate (Fru-1,6-P2) and fructose-1-phosphate (Fru-1-P), with a preference for Fru-1,6-P2 over Fru-1-P, but they have different kcat/Km values towards these two substrates; isozyme alpha shows the highest value for Fru-1,6-P2. These isozymes show similarity in optimal pHs, thermal stability, and Km values for both Fru-1,6-P2 and Fru-1-P. They are composed of four identical subunits of 40 kDa, forming a tetramer with a molecular weight of approximately 160 kDa. The three isozymes are different in primary structure only at the carboxyl-terminal region encoded by the respective exon 4. Therefore, this region should be primarily responsible for the distinct characteristics of these isozymes.

Amino Acid Sequence↗
PubMed1995

Copy-dependent and position-independent expression of rat aldolase A gene.

In order to understand the molecular mechanisms of the temporal and spatial differences of gene expression in higher organisms, rat aldolase A gene carrying two distinct promoters was introduced into fertilized eggs and the resulting transgenic mice were analyzed. The transgene expression is tissue-specific and is developmentally regulated. In addition, the expression is regulated in a copy-dependent manner irrespective of where the transgene is integrated, suggesting that a mechanism excluding the effect of the integration site exists within the transgene itself. To explore the conformational change of this gene in the genome, the DNase I hypersensitive sites of the gene were examined. Three sites (DHS-1,2, and 3) were identified upstream and downstream of the gene and these sites were retained in the transgene as well as in the gene observed endogenously.

Animals↗
PubMed2003

Purification and characterization of class-I and class-II fructose-1,6-bisphosphate aldolases from the cyanobacterium Synechocystis sp. PCC 6803.

The whole genome sequence database for Synechocystis sp. PCC 6803 has revealed the presence of genes encoding class-I (CI) and class-II (CII) fructose-1,6-bisphosphate aldolases (FBAs) in this organism. Two types of FBA from Synechocystis sp. PCC 6803 were separated by chromatography on phenyl-Sepharose. The activity of the enzyme in the major peak was inhibited by the presence of 25 mM EDTA; however, the activity in the minor peak was not. Therefore, the FBA in the former fractions was designated as CII-FBA, and in the latter designated as CI-FBA. CI-FBA was functionally redundant in Synechocystis sp. PCC 6803, while no disruptant for the gene encoding CII-FBA was obtained under photoautotrophic conditions. The kinetic parameters of CI- and CII-FBAs purified from Synechocystis sp. PCC 6803 in the cleavage reaction of FBP were generally similar, except in their reactivity for SBP. The SBP/FBP activity ratio of the CII-FBA was two times higher than that of the CI-FBA.

Cyanobacteria↗
PubMed1996

A lysine to arginine substitution at position 146 of rabbit aldolase A changes the rate-determining step to Schiff base formation.

Lys146 of rabbit aldolase A [D-fructose-1,6-bis(phosphate): D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13] was changed to arginine by site-directed mutagenesis. The kcat of the resulting mutant protein, K146R, was 500 times slower than wild-type in steady-state kinetic assays for both cleavage and condensation of fructose-1,6-bis(phosphate), while the K(m) for this substrate was unchanged. Analysis of the rate of formation of catalytic intermediates showed K146R was significantly different from the wild-type enzyme and other enzymes mutated at this site. Single-turnover experiments using acid precipitation to trap the Schiff base intermediate on the wild-type enzyme failed to show a build-up of this intermediate on K146R. However, K146R retained the ability to form the Schiff base intermediate as shown by the significant amounts of Schiff base intermediate trapped with NaBH4. In the single-turnover experiments it appeared that the Schiff base intermediate was converted to products more rapidly than it was produced. This suggested a maximal rate of Schiff base formation of 0.022 s-1, which was close to the value of kcat for this enzyme. This observation is strikingly different from the wild-type enzyme in which Schiff base formation is > 100 times faster than kcat. For K146R it appears that steps up to and including Schiff base formation are rate limiting for the catalytic reaction. The carbanion intermediate derived from either substrate or product, and the equilibrium concentrations of covalent enzyme-substrate intermediates, were much lower on K146R than on the wild-type enzyme. The greater bulk of the guanidino moiety may destabilize the covalent enzyme-substrate intermediates, thereby slowing the rate of Schiff base formation such that it becomes rate limiting. The K146R mutant enzyme is significantly more active than other enzymes mutated at this site, perhaps because it maintains a positively charged group at an essential position in the active site or perhaps the Arg functionally substitutes as a general acid/base catalyst in both Schiff base formation and in subsequent abstraction of the C4-hydroxyl proton.

Animals↗
PubMed2004

A thermostable variant of fructose bisphosphate aldolase constructed by directed evolution also shows increased stability in organic solvents.

Thermostable variants of the Class II fructose bisphosphate aldolase have been isolated following four rounds of directed evolution using DNA shuffling of the fda genes from Escherichia coli and Edwardsiella ictaluri. Variants from all four generations of evolution have been purified and characterized. The variants show increased thermostability with no loss of catalytic function at room temperature. The temperature at which 50% of the initial enzyme activity is lost after incubation for 10 min (T50) of the most stable variant, 4-43D6, is increased by 11-12 degrees C over the wild-type enzymes and the half-life of activity at 53 degrees C is increased approximately 190-fold. In addition, variant 4-43D6 shows increased stability to treatment with organic solvents. DNA sequencing of the evolved variants has identified the mutations which have been introduced and which lead to increased thermostability, and the role of the mutations introduced is discussed.

Acetonitriles↗
PubMed1981

Chemistry of proton abstraction by glycolytic enzymes (aldolase, isomerases and pyruvate kinase).

Intermediates have been synthesized that are rapidly utilized by triose phosphate isomerase, yeast aldolase and pyruvate kinase. In each case the compounds have the properties of an enol expected for a stepwise proton transfer mechanism. Apparently the apparatus required for doing this chemistry is sufficiently unique for a large measure of structural homology to have been imposed upon the enzymes of this class during evolution.

Biological Evolution↗
PubMed1981

The low-resolution structure of human muscle aldolase.

The three-dimensional structure of human muscle aldolase has been solved at 5 A resolution with the use of two isomorphous heavy atom derivatives. The enzyme's four subunits are arranged about three mutually perpendicular intersecting twofold axes to form a compact spherical molecule. The subunit boundaries are clearly defined but a possible domain structure is not apparent in this preliminary electron density map.

Binding Sites↗