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PubMed1993

Purification and properties of the physically associated meta-cleavage pathway enzymes 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating) from Pseudomonas sp. strain CF600.

The final two steps in the dmp operon-encoded meta-cleavage pathway for phenol degradation in Pseudomonas sp. strain CF600 involve conversion of 4-hydroxy-2-ketovalerate to pyruvate and acetyl coenzyme A (acetyl-CoA) by the enzymes 4-hydroxy-2-ketovalerate aldolase and aldehyde dehydrogenase (acylating) [acetaldehyde:NAD+ oxidoreductase (CoA acetylating), EC 1.2.1.10]. A procedure for purifying these two enzyme activities to homogeneity is reported here. The two activities were found to copurify through five different chromatography steps and ammonium sulfate fractionation, resulting in a preparation that contained approximately equal proportions of two polypeptides with molecular masses of 35 and 40 kDa. Amino-terminal sequencing revealed that the first six amino acids of each polypeptide were those deduced from the previously determined nucleotide sequences of the corresponding dmp operon-encoded genes. The isolated complex had a native molecular mass of 148 kDa, which is consistent with the presence of two of each polypeptide per complex. In addition to generating acetyl-CoA from acetaldehyde, CoA, and NAD+, the dehydrogenase was shown to acylate propionaldehyde, which would be generated by action of the meta-cleavage pathway enzymes on the substrates 3,4-dimethylcatechol and 4-methylcatechol. 4-Hydroxy-2-ketovalerate aldolase activity was stimulated by the addition of Mn2+ and, surprisingly, NADH to assay mixtures. The possible significance of the close physical association between these two polypeptides in ensuring efficient metabolism of the short-chain aldehyde generated by this pathway is discussed.

Aldehyde Oxidoreductases↗
PubMed1998

Biochemical and genetic characterization of trans-2'-carboxybenzalpyruvate hydratase-aldolase from a phenanthrene-degrading Nocardioides strain.

trans-2'-Carboxybenzalpyruvate hydratase-aldolase was purified from a phenanthrene-degrading bacterium, Nocardioides sp. strain KP7, and characterized. The purified enzyme was found to have molecular masses of 38 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography. Thus, the homotrimer of the 38-kDa subunit constituted an active enzyme. The Km and kcat values of this enzyme for trans-2'-carboxybenzalpyruvate were 50 microM and 13 s(-1), respectively. trans-2'-Carboxybenzalpyruvate was transformed to 2-carboxybenzaldehyde and pyruvate by the action of this enzyme. The structural gene for this enzyme was cloned and sequenced; the length of this gene was 996 bp. The deduced amino acid sequence of this enzyme exhibited homology to those of trans-2'-hydroxybenzalpyruvate hydratase-aldolases from Pseudomonas putida PpG7 and Pseudomonas sp. strain C18.

Actinomycetales↗
PubMed2003

Characterization of the 4-carboxy-4-hydroxy-2-oxoadipate aldolase gene and operon structure of the protocatechuate 4,5-cleavage pathway genes in Sphingomonas paucimobilis SYK-6.

The protocatechuate (PCA) 4,5-cleavage pathway is the essential metabolic route for degradation of low-molecular-weight products derived from lignin by Sphingomonas paucimobilis SYK-6. In the 10.5-kb EcoRI fragment carrying the genes for PCA 4,5-dioxygenase (ligAB), 2-pyrone-4,6-dicarboxylate hydrolase (ligI), 4-oxalomesaconate hydratase (ligJ), and a part of 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (ligC), we found the ligK gene, which encodes 4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolase. The ligK gene was located 1,183 bp upstream of ligI and transcribed in the same direction as ligI. We also found the ligR gene encoding a LysR-type transcriptional activator, which was located 174 bp upstream of ligK. The ligK gene consists of a 684-bp open reading frame encoding a polypeptide with a molecular mass of 24,131 Da. The deduced amino acid sequence of ligK showed 57 to 88% identity with those of the corresponding genes recently reported in Sphingomonas sp. strain LB126, Comamonas testosteroni BR6020, Arthrobacter keyseri 12B, and Pseudomonas ochraceae NGJ1. The ligK gene was expressed in Escherichia coli, and the gene product (LigK) was purified to near homogeneity. Electrospray-ionization mass spectrometry indicated that LigK catalyzes not only the conversion of CHA to pyruvate and oxaloacetate but also that of oxaloacetate to pyruvate and CO(2). LigK is a hexamer, and its isoelectric point is 5.1. The K(m) for CHA and oxaloacetate are 11.2 and 136 micro M, respectively. Inactivation of ligK in S. paucimobilis SYK-6 resulted in the growth deficiency of vanillate and syringate, indicating that ligK encodes the essential CHA aldolase for catabolism of these compounds. Reverse transcription-PCR analysis revealed that the PCA 4,5-cleavage pathway genes of S. paucimobilis SYK-6 consisted of four transcriptional units, including the ligK-orf1-ligI-lsdA cluster, the ligJAB cluster, and the monocistronic ligR and ligC genes.

Aldehyde-Lyases↗
PubMed2004

Presence of a novel phosphopentomutase and a 2-deoxyribose 5-phosphate aldolase reveals a metabolic link between pentoses and central carbon metabolism in the hyperthermophilic archaeon Thermococcus kodakaraensis.

Numerous bacteria and mammalian cells harbor two enzymes, phosphopentomutase (PPM) and 2-deoxyribose 5-phosphate aldolase (DERA), involved in the interconversion between nucleosides and central carbon metabolism. In this study, we have examined the presence of this metabolic link in the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. A search of the genome sequence of this strain revealed the presence of a closely related orthologue (TK2104) of bacterial DERA genes while no orthologue related to previously characterized PPM genes could be detected. Expression, purification, and characterization of the TK2104 protein product revealed that this gene actually encoded a DERA, catalyzing the reaction through a class I aldolase mechanism. As PPM activity was detected in T. kodakaraensis cells, we partially purified the protein to examine its N-terminal amino acid sequence. The sequence corresponded to a gene (TK1777) similar to phosphomannomutases within COG1109 but not COG1015, which includes all previously identified PPMs. Heterologous gene expression of TK1777 and characterization of the purified recombinant protein clearly revealed that the gene indeed encoded a PPM. Both enzyme activities could be observed in T. kodakaraensis cells under glycolytic and gluconeogenic growth conditions, whereas the addition of ribose, 2-deoxyribose, and 2'-deoxynucleosides in the medium did not lead to a significant induction of these activities. Our results clearly indicate the presence of a metabolic link between pentoses and central carbon metabolism in T. kodakaraensis, providing an alternative route for pentose biosynthesis through the functions of DERA and a structurally novel PPM.

Aldehyde-Lyases↗
PubMed1966

Properties of a Mutant of Escherichia coli with a Temperature-sensitive Fructose-1,6-Diphosphate Aldolase.

Böck, August (Purdue University, Lafayette, Ind.), and Frederick C. Neidhardt. Properties of a mutant of Escherichia coli with a temperature-sensitive fructose-1,6-diphosphate aldolase. J. Bacteriol. 92:470-476. 1966.-A mutant of Escherichia coli in which fructose-1,6-diphosphate aldolase functions at 30 C but not at 40 C was used to study the physiological effect of a specific block in the Embden-Meyerhof glycolytic pathway. Growth of the mutant at 40 C was found to be inhibited by the presence of glucose or certain related compounds in the medium. At 40 C, glucose was metabolized at 30 to 40% of the control rate and was abnormal in that glucose was converted into other six-carbon substances (probably gluconate, in large part) that were released into the culture medium. The inhibition was complete, but transient; its duration depended upon the initial amount of inhibitor added. The resumption of growth at 40 C was correlated with the further catabolism of the excreted compounds. When glycerol was used to grow the mutant at 40 C, the growth inhibition by glucose was accompanied by cessation of glycerol metabolism. Growth on alpha-glycerol phosphate was not inhibited under these conditions, implicating glycerol kinase as a possible site of inhibition; no inhibition of glycerol kinase by sugar phosphates, however, could be detected in vitro. The inhibitory effect of glucose on growth at 40 C is not caused by a deficit of intracellular adenosine triphosphate, but may be the result of a generalized poisoning of many cell processes by a greatly increased intracellular concentration of fructose-1,6-diphosphate, the substrate of the damaged enzyme.

Journal Article↗
PubMed1966

Serum aldolase and phosphocreatine kinase in umbilical cord blood.

Aldolase was estimated in the cord blood of 81 newborn infants and phosphocreatine kinase in 87 infants. There is a wide range in the results, with some values falling in the range reported in children with muscular dystrophy or of carriers of the disease. There is no correlation of the serum enzyme levels with the infant's birth weight. High levels of phosphocreatine kinase were found in infants of mothers with pre-eclamptic toxaemia. A single estimation of cord phosphocreatine kinase and aldolase is of little help in determining whether or not an infant has muscular dystrophy.

Journal Article↗
PubMed1975

The sterochemistry at carbon 3 of pyruvate lyase condensation products. 2-Keto-3-deoxygluconate 6-phosphate and 2-keto-3-deoxygalactonate-6-phosphate aldolase of Pseudomonas saccharophila.

In Pseudomonas saccharophila 2-keto-3-deoxygalactonate-6-P aldolase (EC 4.1.2.21) is induced by growth on galatose while 2-keto-3-deoxygluconate-6-P aldolase (EC 4.1.2.14) is constitutive. These enzymes catalyze identical reactions except for the configuration fixed at C-4 during the condensation reaction. It was found with each enzyme that in a condensation between [3-3H3]pyruvate and D-glyceraldehyde-3-P, the respective condensation products were formed 8 to 10 times faster than tritium was released to water. Since pyruvate deprotonation is obligatory for condensation, the above result requires a hydrogen isotope effect in enolpyruvate formation, which must be then at least partially rate limiting for C--C synthesis. Further, condensation between D-glyceraldehyde-3-P and (3R)-[3-3H, 2H,H]pyruvate or (3S)-[3-3H, 2H,H]pyruvate, as catalyzed by each enzyme, enriched for (3R)- and (3S)-3-3H, 2H-labeled condensation product, respectively. Thus, each enzyme catalyzes C--C and C--H synthesis with retention of configuration at C-3. This shows that the active sites of both enzymes are asymmetric since solutes can only approach a single face of the bound pyruvyl enolate. In addition, the respective aldehyde specific portions of the two active sites must have opposite chiralities, with respect to each other, for correctly orienting the carbonyl faces of the incoming D-glyceraldehyde-3-P, to generate the correct configuration at C-4 of the respective condensation products.

Aldehyde-Lyases↗
PubMed2001

Co-expression of Triosephosphate Isomerase, Fructose-1, 6-bisphosphate Aldolase and Fructose-1, 6-bisphosphatase in E.coli.

To establish a way to control or to decrease the daily increasing concentration of atmospheric CO(2), metabolically engineering Cyanobacteria was taken for the improvement of its efficiency of photosynthetic CO(2) fixation. As a preliminary stage of this study, three genes coding for three important Calvin cycle enzymes, i.e. triosephosphate isomerase (TPI), fructose-1, 6-bisphosphate aldolase(FBP aldolase),and fructose-1, 6-bisphosphatase(FBPase), respectively, have been cloned into one plasmid, pTrcFAT, which is controlled by promoter trc. Successful co-transcriptional expression of these three genes resulted inhigh yields of these enzymes under the induction of 0.25 mmol/L IPTG. Bioassay showed that the expressed enzymes from one liter of culture could directly catalyze DHAP conversion into 700 &mgr;mol of fructose-6-phosphate (F-6-P) per one minute. Furthermore, in order to introduce the three genes co-expression system into Cyanobacteria, a shuttle plasmid between E.coli and Cyanobacteria was constructed using plasmid pTrcFAT and a shuttle vector pDC-8, forming ashuttle plasmid pDCFAT-2 containing a dimer of the three genes co-expression operator. Successful co-expression in E.coli of pDCFAT-2 with higher full activity has been obtained. This shuttle was used to transform of Cyanobacteria Synechococcus sp. PCC 7942, and a few positive colonies were obtained.

Journal Article↗
PubMed1997

Cloning and High Expression in E.coli of a Chimeric Gene Coding for Rice Fructose-l,6-bisphosphate Aldolase.

A chimeric gene (l 104 bp) coding for rice fructose-l,6-bisphosphate aldolase has been constructed by DNA recombination of a synthetic 5'- fragment (-24 to 272) and an RT-PCR amplified product at restriction site S fu I. The synthetic fragment was assembled from six oligonucleotides by T4 DNA ligase reaction according to a single-stranded DNA method (Chen H-B et al, Nucleic Acids Res 1990, 18, 871-878), the PCR amplified fragment (217 - 1 080) was obtained by carrying out a PCR in the presence of rice cDNA as the template and both the 5'- and the 3'- primers. The whole gene was successively cloned into plasmids pWR13 and pPLc2833, and highly expressed in E. coli to produce the expected product. After purification through stepwise precipitation and cation-exchange column chromatography, the recombinant aldolase showed an enzyme activity as high as (1l.0 +/- 0.3) units/mg with the turnover number = 27s(-1) and K(m) (FBP)=4.2 &mgr;M by two different methods.

Journal Article↗
PubMed1990

Active-site residues of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli. Bromopyruvate inactivation and labeling of glutamate 45.

Treatment of pure 2-keto-4-hydroxyglutarate aldolase from Escherichia coli, a "lysine-type," Schiff-base mechanism enzyme, with the substrate analog bromopyruvate results in a time- and concentration-dependent loss of enzymatic activity. Whereas the substrates pyruvate and 2-keto-4-hydroxyglutarate provide greater than 90% protection against inactivation by bromopyruvate, no protective effect is seen with glycolaldehyde, an analog of glyoxylate. Inactivation studies with [14C] bromopyruvate show the incorporation of 1.1 mol of 14C-labeled compound/enzyme subunit; isolation of a radioactive peptide and determination of its amino acid sequence indicate that the radioactivity is associated with glutamate 45. Incubation of the enzyme with excess [14C]bromopyruvate followed by denaturation with guanidine.HCl allow for the incorporation of carbon-14 at cysteines 159 and 180 as well. Whereas the presence of pyruvate protects Glu-45 from being esterified, it does not prevent the alkylation of these 2 cysteine residues. The results indicate that Glu-45 of E. coli 2-keto-4-hydroxyglutarate aldolase is essential for catalytic activity, most likely acting as the amphoteric proton donor/acceptor that is required as a participant in the overall mechanism of the reaction catalyzed.

Affinity Labels↗
PubMed1980

Lactose and D-galactose metabolism in Staphylococcus aureus. IV. Isolation and properties of a class I D-ketohexose-1,6-diphosphate aldolase that catalyzes the cleavage of D-tagatose 1,6-diphosphate.

The inducible D-ketohexose-1,6-diphosphate aldolase that functions in the metabolism of lactose and D-galactose in Staphylococcus aurues was purified to electrophoretic homogeneity from an extract of D-galactose-grown cells. At saturating substrate concentrations, D-tagatose 1,6-diphosphate was cleaved to dihydroxyacetone phosphate plus D-glyceraldehyde 3-phosphate at twice the rate of D-fructose 1,6-diphosphate; Km values for D-tagatose 1,6-diphosphate and D-fructose 12,6-diphosphate were 1.5 mM and 2.5 mM, respectively. The enzyme catalyzed the aldol condensation of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate to yield a mixture of the 1,6-diphosphate derivatives of D-tagatose, D-fructose, D-sorbose, and D-psicose, indicating that it also catalyzes the cleavage of all four D-2-ketohexose 1,6-diphosphates. The enzyme was not inhibited by EDTA and it had no divalent metal ion requirement, but it did exhibit substrate-dependent inactivation by NaBH4, indicating that it is a Class I (Schiff's base) aldolase. Density gradient centrifugation and gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme exists as a monomer with amolecular weight of about 37,000 and a sedimentation coefficient of 3.4 S. Data on the stability, pH optimum, and inducibility of the enzyme are also presented.

Aldehyde-Lyases↗
PubMed2000

An efficient benchtop system for multigram-scale kinetic resolutions using aldolase antibodies.

The preparative scale kinetic resolution of racemic aldols 1-4 using aldolase antibodies 38C2 (Aldrich no. 47995-0) and 84G3 (Aldrich no. 52785-8) is described. These reactions use a biphasic aqueous/organic solvent system that allows the catalyst to be reused. Reaction scales range from miligrams to grams, with 0.0086 to 0.12 mol% of antibody binding sites. Because antibodies 38C2 and 84G3 have opposite enantioselectivities, both aldol product enantiomers are accessible by kinetic resolution.

Aldehydes↗
PubMed1995

Developmental alteration of the chromatin state at promoter/replication origin region of the aldolase B locus precedes transcriptional activation in the liver.

Liver-specific expression of the rat aldolase B (AldB) gene is conferred by proximal promoter region (-200 bp to + 1 bp), which is centered on an origin region of DNA replication. Transcriptional activation of the gene in the liver occurs during the late one-third of fetal stage. To know the mechanism involved in such activation, we studied developmental changes in chromatin structure and in the extent of CpG methylation in the promoter/origin region of the gene. At an early fetal stage, when the AldB gene in the liver is not yet activated, the gene chromatin had two DNase 1-hypersensitive sites in the promoter region. One corresponded to that typical of AldB-expressing cells in the adult. The other, located approximately 200 bp upstream of the above site, disappeared as the activation of transcription started. A CpG dinucleotide in the promoter/origin region was heavily methylated at an early stage of gestation, but progressively demethylated as the liver develops. This CpG site is located at the center of an important binding site for a transcription factor. These changes occurred early in the fetal stage, prior to the gene activation, and were thus thought to be associated with differentiation of the liver cell or with cessation of cell proliferation.

Animals↗
PubMed2005

The spectrum of aldolase B (ALDOB) mutations and the prevalence of hereditary fructose intolerance in Central Europe.

We investigated the molecular basis of hereditary fructose intolerance (HFI) in 80 patients from 72 families by means of a PCR-based mutation screening strategy, consisting of heteroduplex analysis, restriction enzyme digest, DNA single strand electrophoresis, and direct sequencing. For a subset of patients mutation screening with DHPLC was established which turned out to be as fast and as sensitive as the more conventional methods. Fifteen different mutations of the aldolase B (ALDOB) gene were identified in HFI patients. As in smaller previous studies, p.A150P (65%), p.A175D (11%) and p.N335K (8%) were the most common mutated alleles, followed by c.360_363delCAAA, p.R60X, p.Y204X, and c.865delC. Eight novel mutations were identified in eight families with HFI: a small indel mutation (c.1044_1049delTTCTGGinsACACT), two small deletions (c.345_372del28; c.841_842delAC), two splice site mutations (c.113-1G>A, c.799+2T>A), one nonsense mutation (c.612T>G (p.Y204X)), and two missense mutations (c.532T>C (p.C178R), c.851T>C (p.L284P)). By mutation screening for the three most common ALDOB mutations by DHPLC in 2,000 randomly selected newborns we detected 21 heterozygotes. Based on these data and after correction for less common and private ALDOB mutations, HFI prevalence in central Europe is estimated to be 1:26,100 (95% confidence interval 1: 12,600-79,000).

Chromatography, High Pressure Liquid↗
PubMed1994

Copurification of cytosolic fructose-1,6-bisphosphatase and cytosolic aldolase from endosperm of germinating castor oil seeds.

The cytosolic isozymes of fructose-1,6-bisphosphatase (FBPasec) and aldolase (ALDc) from germinating castor oil seed endosperm (COS) (Ricinus communis L.; cv Hale) were purified to homogeneity and final specific activities 49 and 2.8 (mumol product produced/min)/mg protein, respectively. Nondenaturing polyacrylamide gel electrophoresis of the final FBPasec preparation resolved a single protein-staining band which comigrated with FBPase activity. Two protein-staining bands of 41 and 39 kDa that occurred in an approximate 1:1 ratio were observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final FBPasec preparation. Rabbit anti-(FBPasec) immune serum immunoprecipitated the activities of FBPasec, but not that of the plastidic isozyme of FBPase from germinated COS. Immunoblot analysis utilizing affinity purified anti-(COS FBPasec) immunoglobulin G established that the 39-kDa subunit of FBP-asec did not arise via proteolytic cleavage of the 41-kDa subunit during tissue extraction and enzyme purification. However, FBPasec was susceptible to degradation by endogenous protease(s) during incubation of an acidic (pH 5.9) clarified COS extract at 25 degrees C. This proteolysis caused the production of a 32-kDa antigenic polypeptide and resulted in FBPase inactivation. Gel filtration indicated that purified FBPasec exists in at least 8 different oligomeric forms ranging in size from > 2 million to < 34 kDa. The majority of FBPasec, however, eluted as a 143-kDa heterotetramer. Sodium dodecyl sulfate gel electrophoresis of the final ALDc preparation yielded a single 40-kDa protein-staining polypeptide that cross-reacted with anti-(carrot ALDc) IgG. FBPasec copurified with ALDc through polyethylene glycol fractionation, Q-Sepharose, and phosphocellulose chromatographies, and the intensity of the fluorescence emission spectrum of ALDc was greatly reduced in the presence of COS FBPasec, but not rabbit muscle FBPase. These findings suggest that these two metabolically sequential enzymes might specifically interact in the cytosol of the highly gluconeogenic germinating COS. Our results also demonstrate that endogenous nonspecific acid phosphatase activity can interfere with the spectrophotometric assay for FBPase and can thus result in overestimations of FBPase activity in impure plant extracts.

Ricinus communis↗
PubMed2000

Characterization of the responsive elements to hormones in the rat aldolase B gene.

Transcription of the aldolase B gene, AldB, in the liver is regulated by hormones such as insulin and glucagon. To characterize the elements that are responsive to these hormones in the upstream region of AldB, plasmids carrying various length of the upstream region of this gene were constructed and transfected to primary cultured rat hepatocytes. The promoter activities were gradually increased by progressive deletion of the 5'-upstream region, and high activities were observed for constructs carrying the sequence between -408 and -85 bp, suggesting the presence of suppressive element(s) in the upstream region of -409 bp. The transcription activities of the mutants containing the sequences between -228 and -85 bp were enhanced by insulin, and glucagon suppressed the transcription activities of those containing the sequence between -764 and -85 bp. Two sequence elements similar to the cAMP-responsive element (CRE), one from -89 to -82 bp and another from +13 to +20 bp, were found in the upstream sequence of the gene. The latter element is not functional because its deletion did not affect either the transcription efficiency or glucagon response. However, the deletion of the former element diminished both functions. A gel retardation assay showed that the nuclear factor binds to the former element, which was competitive with authentic CRE oligonucleotide but not with the mutant CRE one. These results suggest that the CRE-like element in the promoter region is prerequisite for both fundamental transcription efficiency of the gene and suppression by glucagon in hepatocytes.

5' Untranslated Regions↗
PubMed1993

Multiple control elements regulate transcription from the most distal promoter of human aldolase A gene.

Three major transcripts, differing in their 5'-untranslated regions, are produced from the human aldolase A gene by alternative usage of three promoters, designated distal, middle and proximal. We report that the genomic region (distal promoter) upstream from the first leader exon (exon L1) efficiently directs transcription of a reporter CAT gene after transient transfections in human hepatoma cells (Hep3B) in a fashion independent from the other two promoters of the same gene. The distal promoter region contains cis-acting elements that regulate transcription both positively and negatively.

Base Sequence↗