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PubMed1983

A radioisotopic method for fructose-1-phosphate aldolase assay that facilitates diagnosis of hereditary fructose intolerance.

A sensitive new method in which D-[U-14C]fructose-1-phosphate is used for fructose-bisphosphate aldolase (EC 2.1.2.13) assay is described. The radioactive fructose-1-phosphate compound was prepared from [U-14C]fructose by use of partly purified fructokinase (EC 2.7.1.4). With this method we measured normal values for aldolase in human liver (2.4-10.0 nmol/min per mg of protein), kidney (3.6-3.8), and intestine (4.2-10.0) as well as Km values for fructose-1-phosphate (approximately 1.0-2.2 mmol/L). In patients with hereditary fructose intolerance the aldolase activity in liver and intestine was less than 10% of normal values. The Lineweaver-Burk plots for data from patients with hereditary fructose intolerance were hyperbolic, indicating a structural alteration in the enzyme.

Adult↗
PubMed1980

[Localization of embryonic aldolase isoenzyme in rat liver cells after a single injection of the carcinogen, 4-dimethylaminoazobenzene, and its noncarcinogenic analog].

The localization of a fetal isoenzyme of aldolase (A) in rat liver cells early after a single injection of carcinogen 4-dimethylaminoazobenzene and its noncarcinogenic analog 4-diethylaminoazobenzene has been studied using the immunofluorescent method. Aldolase A was found in the cytoplasm of oval and "transition" cells. These cells appeared in rat liver as a result of treatment with carcinogen and its analog. In mature hepatocytes aldolase A was not found either in intact rat liver, after the treatment with carcinogen or its analog.

Animals↗
PubMed1980

[The value determination of serum aldolase for findings of metastases and relapses on bronchial carcinoma (author's transl)].

In 180 patients with histologically confirmed bronchial carcinoma exactitude of aldolase as a means for carrying out a screening test for metastases and relapses was checked. Enzyme activity was mostly normal in patients with limited extended local tumor growth including regional metastases as well as in tumour relapses. The group of patients with distant metastases, showed a distribution of increases and normal enzyme values which was equal; when differentiating further the majority of pathological aldolase values (79.4 per cent) was found in the group with liver metastases. We therefore regard as valuable the determination of serum aldolase when locking for liver metastases in confirmed bronchial carcinoma.

Adult↗
PubMed1980

[Thermal sensitivity of aldolase isoenzymes in the organs of cattle, sheep and swine].

During the investigation of homogenates from organs of cattle, sheep and swine (a heart, a kidney, a pancreas, a liver, a thigh muscle, a spleen, a lung) the highest activity of aldolase was proved in the bodily muscles, whereas in the other organs it did not show great differences. Thus it is determined as an ubiquitous enzyme in all these kinds of farm animals. After heating at 61 degrees C for 15 min. aldolase from the bodily muscles of cattle, sheep and swine, showed a high degree of thermostability (residual activity of 86 = 92%), whereas in the parenchymatous organs the enzyme was considerably thermosensitive (residual activity of 15.2--48.4%). The liver isoenzyme of aldolase showed the highest degree of thermosensitivity and had a residual activity of hardly 8--12%. A viewpoint is endorsed, namely that on that basis can be differentiated some pathological processes in the musculature from the pathological processes in the parenchymatous organs.

Animals↗
PubMed1993

Site-directed mutagenesis identifies aspartate 33 as a previously unidentified critical residue in the catalytic mechanism of rabbit aldolase A.

The expression and purification of the rabbit muscle aldolase A (D-fructose 1,6-bisphosphate:D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) from an expression plasmid in bacteria is described. The enzyme is produced in bacteria at a level of 300 mg/liter and is indistinguishable from the enzyme isolated from muscle in assays using fructose 1,6-bisphosphate and fructose 1-phosphate. The recombinant enzyme has the same primary, secondary, and quaternary structure as the muscle enzyme. Aspartic acid 33, found near the active site lysine in the crystal structure, is changed to alanine, serine, and glutamic acid by site-directed mutagenesis, resulting in the mutant proteins, D33A, D33S, and D33E, respectively. The mutant enzymes are purified by substrate affinity elution from carboxylmethyl-Sepharose, the same method as that used for the wild-type enzyme. The secondary and quaternary structure of D33A is identical to wild-type aldolase when analyzed by light scattering, gel filtration, and circular dichroism. Moreover, the hexose substrate can be fixed in the active site by reduction of the Schiff base with sodium borohydride, indicating that the active site is not drastically altered. These single mutations in the active site have a serious effect on the activity of the enzyme. In addition, the rate of carbanion oxidation for D33A is 17-29 times slower when the substrate is fructose 1,6-bisphosphate versus dihydroxyacetone phosphate, whereas in the wild-type there is no significant difference in these rates. This evidence and the conservation of this residue in other class I aldolases indicate that aspartic acid 33 is an essential residue in the catalytic mechanism, possibly involved in abstraction of the carbon 4 hydroxyl proton.

Amino Acid Sequence↗
PubMedDate not supplied

[Conformational changes in rabbit muscle aldolase under normal conditions and following prolonged starvation].

The experiments were aimed at studying conformation differences of muscle aldolase in normal rabbits and those fasting for a long time and at finding the areas of polypeptide chains affected by the changes. For this purpose the difference, temperature- and solvent-perturbation spectra of the compared proteins were examined. On the basis of the data obtained on the same amount of chromophore groups in both proteins a conclusion is drawn on a more hydrophobic surrounding of tyrosine and tryptophan residues in aldolase of the long-fasting animals. When determing the content of the tyrosine and tryptophan surface residues an increase (from 13 to 21) was found in the number of the tyrosine residues in aldolase of the long-fasting animals. An assumption is advanced on localization of the primary structure changes in the molecule sites rich in chromophore groups or those located closer to them.

Animals↗
PubMed1998

Cloning and characterization of Schistosoma mansoni fructose-1,6-bisphosphate aldolase isoenzyme.

A Schistosoma mansoni cercarial cDNA expression library, constructed in lambda gt11, was screened using the IgG fraction of sera taken from rabbits vaccinated with irradiated cercariae. A positive cDNA clone (1,431 base pairs) was selected and characterized. The amino acid sequence predicted from the cDNA sequence identified a polypeptide of 363 amino acids that showed significant homology to different family members of the enzyme fructose-1,6-bisphosphate aldolase (EC 1.4.2.13). The identity was 66% and 65% with human C and A isoenzymes, respectively. Active sites and substrate-binding determinant analysis suggest that the isolated enzyme in terms of function resembles type A aldolase. The recombinant protein expressed in the vector pGEX-2T was found to be active enzymatically. Antibodies raised against the purified recombinant protein recognized a 40-kDa band in extracts from cercariae, schistosomula (5 and 25 days), adult worms, and eggs. Using immunocytochemistry, aldolase localized to the tegumental region of the adult worms.

Amino Acid Sequence↗
PubMed2000

Fluorogenic polypropionate fragments for detecting stereoselective aldolases.

A series of fluorogenic polypropionate fragments has been prepared. These undergo retroaldolization to an intermediate aldehyde that liberates the fluorescent product umbelliferone by a secondary beta-elimination reaction. leading to a >20-fold increase in fluorescence (lambda(em) = 460 +/- 20 nm, lambdaex = 360 +/- 20 nm). By applying the principle of microscopic reversibility to the reversible aldol reaction, we can use these substrates to detect stereoselective aldolases. Test substrates are available to probe the classical cases of syn- and anti-selective aldolization (11a-d), Cram/ anti-Cram-selective aldolization (10a-d), and double stereoselective aldolization (3a-h). The selectivity of aldolase antibody 38C2 for these substrates is demonstrated as an example. The assay is suitable for high-throughput screening for catalysis in microtiter plates, and therefore provides a convenient tool for the isolation of new stereoselective aldolases from catalyst libraries.

Aldehyde-Lyases↗
PubMed2006

Directed evolution of an industrial biocatalyst: 2-deoxy-D-ribose 5-phosphate aldolase.

Aldolases are emerging as powerful and cost efficient tools for the industrial synthesis of chiral molecules. They catalyze enantioselective carbon-carbon bond formations, generating up to two chiral centers under mild reaction conditions. Despite their versatility, narrow substrate ranges and enzyme inactivation under synthesis conditions represented major obstacles for large-scale applications of aldolases. In this study we applied directed evolution to optimize Escherichia coli 2-deoxy-D-ribose 5-phosphate aldolase (DERA) as biocatalyst for the industrial synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside. This versatile chiral precursor for vastatin drugs like Lipitor (atorvastatin) is synthesized by DERA in a tandem-aldol reaction from chloroacetaldehyde and two acetaldehyde equivalents. However, E. coli DERA shows low affinity to chloroacetaldehyde and is rapidly inactivated at aldehyde concentrations useful for biocatalysis. Using high-throughput screenings for chloroacetaldehyde resistance and for higher productivity, several improved variants have been identified. By combination of the most beneficial mutations we obtained a tenfold improved variant compared to wild-type DERA with regard to (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside synthesis, under industrially relevant conditions.

Aldehyde-Lyases↗
PubMed2006

Influence of secondary reactions on the synthetic efficiency of DHAP-aldolases.

The effect of secondary reactions on DHAP-dependent aldolase stereoselective synthesis yields is reported. The fuculose-1-phosphate aldolase catalyzed synthesis between DHAP and Cbz-S-alaninal has been chosen as case study. It has been demonstrated that DHAP is not only chemically degraded in the reaction medium, but also enzymatically. The last reaction has been shown to take place when type II aldolases are used as biocatalysts. In order to minimize the effect of non-desired reactions, temperature reduction has been shown to be favorable, and operation at 4 degrees C has been chosen as appropriate. On the other hand, the fed-batch addition of DHAP also increased the synthesis yields and, combined with low temperature, led to almost quantitative conversion.

Alanine↗
PubMed2003

Stereoselective aldol additions catalyzed by dihydroxyacetone phosphate-dependent aldolases in emulsion systems: preparation and structural characterization of linear and cyclic iminopolyols from aminoaldehydes.

The potential of dihydroxyacetone phosphate (DHAP)-dependent aldolases to catalyze stereoselective aldol additions is, in many instances, limited by the solubility of the acceptor aldehyde in aqueous/co-solvent mixtures. Herein, we demonstrate the efficiency of emulsion systems as reaction media for the class I fructose-1,6-bisphosphate aldolase (RAMA) and class II recombinant rhamnulose-1-phosphate aldolase from E. coli (RhuA)-catalyzed aldol addition between DHAP and N-benzyloxycarbonyl (N-Cbz) aminoaldehydes. The use of emulsions improved the RAMA-catalyzed aldol conversions by three to tenfold relative to those in conventional DMF/water mixtures. RhuA was more reactive than RAMA towards the N-Cbz aminoaldehydes regardless of the reaction medium. With (S)- or (R)-Cbz-alaninal, RAMA exhibited preference for the R enantiomer, while RhuA had no enantiomeric discrimination. The linear N-Cbz aminopolyols thus obtained were submitted to catalytic intramolecular reductive amination to afford the corresponding iminocyclitols. This reaction was diastereoselective in all cases examined; the face selectivity was controlled by the stereochemistry of the newly formed hydroxyl group originating from the aldehyde. Characterization of the resulting iminocyclitols allowed the assessment of the diastereoselectivity of the enzymatic aldol reactions with respect to the N-protected aminoaldehyde. RAMA formed single diastereoisomers from N-Cbz-glycinal and from both enantiomers of N-Cbz-alaninal, while 14 % of the epimeric product was observed from N-Cbz-3-aminopropanal. Diastereoselectivity from RhuA was lower than that observed from RAMA. Interestingly, a single diastereoisomer was formed from (S)-Cbz-alaninal, whereas only a 34 % diastereomeric excess was observed from its enantiomer (i.e., (R)-Cbz-alaninal).

Journal Article↗
PubMed2000

Gene cloning and overproduction of low-specificity D-threonine aldolase from Alcaligenes xylosoxidans and its application for production of a key intermediate for parkinsonism drug.

The dtaAX gene encoding a pyridoxal 5'-phosphate (pyridoxal-P)-dependent low-specificity D-threonine aldolase was cloned from the chromosomal DNA of Alcaligenes xylosoxidans IFO 12669. It contains an open reading frame consisting of 1,134 nucleotides corresponding to 377 amino acid residues. The predicted amino acid sequence displayed 54% identity with that of D-threonine aldolase from gram-positive bacteria Arthrobacter sp. DK-38, but showed no significant similarity with those of other known pyridoxal-P enzymes. This gram-negative bacterial enzyme was highly overproduced in recombinant Escherichia coli cells, and the specific activity of the enzyme in the cell extract was as high as 18 U/mg (purified enzyme 38.6 U/mg), which was 6,000 times higher than that from the wild-type Alcaligenes cell extract. The recombinant enzyme was thus feasibly purified to homogeneity by ammonium sulfate fractionation and DEAE-Toyopearl chromatography steps. The recombinant low-specificity D-threonine aldolase was shown to be an efficient biocatalyst for resolution of L-beta-3,4-methylenedioxyphenylserine, an intermediate for production of a therapeutic drug for Parkinson's disease.

Alcaligenes↗
PubMed1983

Dimerization occurs during the reversible acid inactivation of 2-oxo-4-hydroxyglutarate aldolase from Escherichia coli.

Exposure of Escherichia coli 2-oxo-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate glyoxylate-lyase, EC 4.1.3.16) (molecular weight = 63 000) to phosphoric acid at pH 1.6 for 10 min at 4 degrees C causes 95% or greater inactivation. No significant effect on the rate or extent of inactivation is caused by varied aldolase concentrations or the presence of exogenous proteins. Chloride ion (50-100 mM) or 10 mM 2-oxo-4-hydroxyglutarate markedly decreases both the rate and extent of inactivation; good protection is also afforded by 10 mM pyruvate, glyoxylate, glyoxal, 2-oxoglutarate or 2-oxobutyrate. Whereas native aldolase has two free and three buried sulfhydryl groups, all five are exposed in the acid-inactivated enzyme and the molecular weight of this species at pH 1.6 is 126 000. Ultraviolet absorbance difference spectra, circular dichroism spectra and ultracentrifugation studies establish that the inactivation process is characterized by an alteration of secondary and tertiary structure as well as an aggregation to a dimer of the native molecule. Reactivation of enzyme activity to 60-80% of the original level is seen within 20 min at pH 6 to 8; examination of inactivation/reactivation as a function of pH indicates that these two processes occur via kinetically distinct pathways. Native and reactivated enzymes are identical in molecular weight, sulfhydryl titer, Km and alpha-helix content.

Chlorides↗
PubMed2004

Slow-binding inhibition of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase.

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase is a key enzyme in the Entner-Doudoroff pathway of bacteria. It catalyzes the reversible production of KDPG from pyruvate and D-glyceraldehyde 3-phosphate through a class I Schiff base mechanism. On the basis of aldolase mechanistic pathway, various pyruvate analogues bearing beta-diketo structures were designed and synthesized as potential inhibitors. Their capacity to inhibit aldolase catalyzed reaction by forming stabilized iminium ion or conjugated enamine were investigated by enzymatic kinetics and UV-vis difference spectroscopy. Depending of the substituent R (methyl or aromatic ring), a competitive or a slow-binding inhibition takes place. These results were examined on the basis of the three-dimensional structure of the enzyme.

Aldehyde-Lyases↗
PubMed1997

An efficient process for production of N-acetylneuraminic acid using N-acetylneuraminic acid aldolase.

N-acetyl-D-neuraminic acid (Neu5Ac) aldolase (EC 4.1.3.3) has bee reported for synthesis of Neu5Ac,1-5 but there are no reports of processes which do not have significant drawbacks for large-scale operation. Here, Neu5Ac aldolase from an overexpressing recombinant strain of Escherichia coli has been used to develop an immobilized enzyme process for production of Neu5Ac. The enzyme was immobilized onto Eupergit-C and could be reused many times in the reaction. Base-catalyzed epimerization of N-acetyl-D-glucosamine (GlcNAc) yielded GlcNAc/N-acetyl-D-mannosamine (ManNAc) mixtures (c 4:1) which could be used directly in the aldolase reaction; however, inhibition of the enzyme by GlcNAc limited the concentration of ManNAc which could be used in the reaction by this approach. This necessitated the addition of a large molar excess of pyruvate (five- to seven-fold) to drive the equilibrium over to Neu5Ac; nevertheless, a method has been developed to remove the excess pyruvate effectively by complexation with bisulfite, thus allowing Neu5Ac to be recovered by absorption onto an anion-exchange resin. In a second approach, a method has been developed to enrich GlcNAc/ManNAc mixtures for ManNAc. ManNAc can be used at high concentrations in the reaction, thus obviating the need to use a large molar excess of pyruvate. Neu5Ac can be isolated from such reaction mixtures by a simple crystallization. This work shows the importance of integrated process solutions for the effective scale-up of biotransformation reactions.

1-Propanol↗
PubMed2001

Directed evolution of a new catalytic site in 2-keto-3-deoxy-6-phosphogluconate aldolase from Escherichia coli.

BACKGROUND: Aldolases are carbon bond-forming enzymes that have long been identified as useful tools for the organic chemist. However, their utility is limited in part by their narrow substrate utilization. Site-directed mutagenesis of various enzymes to alter their specificity has been performed for many years, typically without the desired effect. More recently directed evolution has been employed to engineer new activities onto existing scaffoldings. This approach allows random mutation of the gene and then selects for fitness to purpose those proteins with the desired activity. To date such approaches have furnished novel activities through multiple mutations of residues involved in recognition; in no instance has a key catalytic residue been altered while activity is retained. RESULTS: We report a double mutant of E. coli 2-keto-3-deoxy-6-phosphogluconate aldolase with reduced but measurable enzyme activity and a synthetically useful substrate profile. The mutant was identified from directed-evolution experiments. Modification of substrate specificity is achieved by altering the position of the active site lysine from one beta strand to a neighboring strand rather than by modification of the substrate recognition site. The new enzyme is different to all other existing aldolases with respect to the location of its active site to secondary structure. The new enzyme still displays enantiofacial discrimination during aldol addition. We have determined the crystal structure of the wild-type enzyme (by multiple wavelength methods) to 2.17 A and the double mutant enzyme to 2.7 A resolution. CONCLUSIONS: These results suggest that the scope of directed evolution is substantially larger than previously envisioned in that it is possible to perturb the active site residues themselves as well as surrounding loops to alter specificity. The structure of the double mutant shows how catalytic competency is maintained despite spatial reorganization of the active site with respect to substrate.

Aldehyde-Lyases↗
PubMed2003

Threonine aldolase and alanine racemase: novel examples of convergent evolution in the superfamily of vitamin B6-dependent enzymes.

Vitamin B(6)-dependent enzymes may be grouped into five evolutionarily unrelated families, each having a different fold. Within fold type I enzymes, L-threonine aldolase (L-TA) and fungal alanine racemase (AlaRac) belong to a subgroup of structurally and mechanistically closely related proteins, which specialised during evolution to perform different functions. In a previous study, a comparison of the catalytic properties and active site structures of these enzymes suggested that they have a catalytic apparatus with the same basic features. Recently, recombinant D-threonine aldolases (D-TAs) from two bacterial organisms have been characterised, their predicted amino acid sequences showing no significant similarities to any of the known B(6) enzymes. In the present work, a comparative structural analysis suggests that D-TA has an alpha/beta barrel fold and therefore is a fold type III B(6) enzyme, as eukaryotic ornithine decarboxylase (ODC) and bacterial AlaRac. The presence of both TA and AlaRac in two distinct evolutionary unrelated families represents a novel and interesting example of convergent evolution. The independent emergence of the same catalytic properties in families characterised by completely different folds may have not been determined by chance, but by the similar structural features required to catalyse pyridoxal phosphate-dependent aldolase and racemase reactions.

Alanine Racemase↗
PubMed1976

Inactivation of bovine liver 2-keto-4-hydroxyglutarate aldolase by cyanide in the presence of aldehydes.

Kinetic data show that the irreversible inactivation of liver 2-keto-4-hydroxyglutarate aldolase observed when the enzyme is incubated with an aldehydic substrate (or substrate analogue) in the presence of cyanide is a biphasic process and can, under certain conditions, involve a direct interaction between the enzyme and cyanide. The kinetic data are consistent with a scheme consisting of three competing reactions: (1) irreversible addition of cyanide to the enzyme-substrate Schiff base intermediate, (2) reversible cyanohydrin formation between cyanide and the aldehydic substrate (or substrate analogue), and (3) an interaction of cyanide with the enzyme which is not substrate dependent. Approximately 0.4 mol of cyanide is associated with 1 mol (120 000 g) of enzyme when 2-keto-4-hydroxyglutarate aldolase is incubated with [14-C]-cyanide followed by exhaustive dialysis; an ionic attachment possibly at a carboxylate binding site, is suggested. Whereas native enzyme, not treated with cyanide, has ten Nbs2-titratable sulfhydryl groups, approximately one less such group reacts with Nbs2 when the aldolase is incubated with cyanide (in the absence of aldehydic substrate). It is suggested that the binding of cyanide results in a conformational change of the enzyme; conformational changes in the presence of cyanide are confirmed by circular dichroism spectra.

Animals↗