Tritium exchange reactions catalyzed by 2-oxo-4-hydroxyglutarate aldolase from Escherichia coli K-12.

Tritiated water and tritiated substrates have been used to study exchange reactions catalyzed by Escherichia coli 2-oxo-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate glyoxylate-lyase, EC 4.1.3.16, 2-oxo-4-hydroxyglutarate in equilibrium pyruvate + glyoxylate). With pyruvate, the enzyme catalyzes a rapid first-order exchange of all three methyl hydrogens in the absence of added acceptor aldehyde (i.e. glyoxylate). This reaction is not rate limiting for aldol condensation or cleavage; quite different pH-activity profiles for the exchange reaction versus aldol cleavage and also comparative effects that pH changes have on Km and V values for the two processes favor this conclusion. The exchange reaction with 2-oxobutyrate, a substrate analog, is stereoselective; one methylene hydrogen is removed at a 6-fold faster rate than the other but eventually both are exchanged. No tritium exchange occurs with glyoxylate.     

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.     

Cloning, nucleotide sequence, overexpression, and inactivation of the Escherichia coli 2-keto-4-hydroxyglutarate aldolase gene.

Having previously determined the complete amino acid sequence of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli (C. J. Vlahos and E. E. Dekker, J. Biol. Chem. 263:11683-11691, 1988), we amplified the gene that codes for this enzyme by the polymerase chain reaction using synthetic degenerate deoxyoligonucleotide primers. The amplified DNA was sequenced by subcloning the polymerase chain reaction products into bacteriophage M13; the nucleotide sequence of the gene was found to be in exact agreement with the amino acid sequence of the gene product. Overexpression of the gene was accomplished by cloning it into the pKK223.3 expression vector so that it was under control of the tac promoter and then using the resultant plasmid, pDP6, to transform E. coli DH5 alpha F'IQ. When this strain was grown in the presence of isopropyl beta-D-thiogalactopyranoside, aldolase specific activity in crude extracts was 80-fold higher than that in wild-type cells and the enzyme constituted approximately 30% of the total cellular protein. All properties of the purified, cloned gene product, including cross-reactivity with antibodies elicited against the wild-type enzyme, were identical with the aldolase previously isolated and characterized. A strain of E. coli in which this gene is inactivated was prepared for the first time by insertion of the kanamycin resistance gene cartridge into the aldolase chromosomal gene.     

Amino acid sequence of the pyruvate and the glyoxylate active-site lysine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase

Pure 2-keto-4-hydroxyglutarate aldolase of Escherichia coli, a "lysine-type" trimeric enzyme which has the unique properties of forming an "abortive" Schiff-base intermediate with glyoxylate (the aldehydic product/substrate) and of showing strong beta-decarboxylase activity toward oxalacetate, binds any one of its substrates (2-keto-4-hydroxyglutarate, pyruvate, or glyoxylate) in a competitive manner. To determine whether the substrates bind at the same or different (juxta-positioned) sites and what degree of homology might exist between the active-site lysine peptide of this enzyme and that of other lysine-type (Class I) aldolases or beta-decarboxylases, the azomethine formed separately by this aldolase with either [14C]pyruvate or [14C]glyoxylate was reduced with CNBH3-. After each enzyme adduct was digested with trypsin, the 14C-labeled peptide was isolated, purified, and subjected to amino acid analysis and sequence determination. In each case, the same 14-amino acid lysine-peptide was isolated and found to have the following primary sequence: Glu-Phe-*Lys-Phe-Phe-Pro-Ala-Glu-Ala-Asn-Gly-Gly-Val-Lys (where * = the active-site lysine). Hence, glyoxylate competes for, and inhibits aldolase activity by reacting with, the one active-site lysine residue/subunit. This active-site lysine peptide has a high degree (65%) of homology with that of 2-keto-3-deoxy-6-phosphogluconate aldolase of Pseudomonas putida but is not similar to that of any Class I fructose-1,6-bisphosphate aldolase or of acetoacetate beta-decarboxylase of Clostridium acetobutylicum. Furthermore, it was found that extensive reaction of glyoxylate with the N-terminal amino group of this enzyme may well be general complicating factor in sequence studies with proteins plus glyoxylate.     

The complete amino acid sequence and identification of the active-site arginine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase

The complete amino acid sequence of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli has been established in the following manner. After being reduced with dithiothreitol, the purified aldolase was alkylated with iodoacetamide and subsequently digested with trypsin. The resulting 19 peptide peaks observed by high performance liquid chromatography, which compared with 21 expected tryptic cleavage products, were all isolated, purified, and individually sequenced. Overlap peptides were obtained by a combination of sequencing the N-terminal region of the intact aldolase and by cleaving the intact enzyme with cyanogen bromide followed by subdigestion of the three major cyanogen bromide peptides with either Staphylococcus aureus V8 endoproteinase, endoproteinase Lys C, or trypsin after citraconylation of lysine residues. The primary structure of the molecule was determined to be as follows. (formula; see text) 2-Keto-4-hydroxyglutarate aldolase from E. coli consists of 213 amino acids with a subunit and a trimer molecular weight of 22,286 and 66,858, respectively. No microheterogeneity is observed among the three subunits. The peptide containing the active-site arginine residue (Vlahos, C. J., Ghalambor, M. A., and Dekker, E. E. (1985) J. Biol. Chem. 260, 5480-5485) was also isolated and sequenced; this arginine residue occupies position 49. The Schiff base-forming lysine residue (Vlahos, C. J., and Dekker, E. E. (1986) J. Biol. Chem. 261, 11049-11055) is located at position 133. Whereas the active-site lysine peptide of this aldolase shows 65% homology with the same peptide of 2-keto-3-deoxy-6-phosphogluconate aldolase from Pseudomonas putida, these two proteins in toto show 49% homology.     

Evidence for an essential arginine residue in the active site of Escherichia coli 2-keto-4-hydroxyglutarate aldolase. Modification with 1,2-cyclohexanedione

Treatment of homogeneous preparations of Escherichia coli 2-keto-4-hydroxyglutarate aldolase with 1,2-cyclohexanedione, 2,3-butanedione, phenylglyoxal, or 2,4-pentanedione results in a time- and concentration-dependent loss of enzymatic activity; the kinetics of inactivation are pseudo-first order. Cyclohexanedione is the most effective modifier; a plot of log (1000/t 1/2) versus log [cyclohexanedione] gives a straight line with slope = 1.1, indicating that one molecule of modifier reacts with each active unit of enzyme. The kinetics of inactivation are first order with respect to cyclohexanedione, suggesting that the loss of activity is due to modification of 1 arginine residue/subunit. Controls establish that this inactivation is not due to modifier-induced dissociation or photoinduced structural alteration of the aldolase. The same Km but decreased Vmax values are obtained when partially inactivated enzyme is compared with native. Amino acid analyses of 95% inactivated aldolase show the loss of 1 arginine/subunit with no significant change in other amino acid residues. Considerable protection against inactivation is provided by the substrates 2-keto-4-hydroxyglutarate and pyruvate (75 and 50%, respectively) and to a lesser extent (40 and 35%, respectively) by analogs like 2-keto-4-hydroxybutyrate and 2-keto-3-deoxyarabonate. In contrast, formaldehyde or glycolaldehyde (analogs of glyoxylate) under similar conditions show no protective effect. These results indicate that an arginine residue is required for E. coli 2-keto-4-hydroxyglutarate aldolase activity; it most likely participates in the active site of the enzyme by interacting with the carboxylate anion of the pyruvate-forming moiety of 2-keto-4-hydroxyglutarate.     

Detection of aldolase activity on polyacrylamide gels: application to 2-keto-4-hydroxyglutarate aldolase.

A method is described for the detection of 2-keto-4-hydroxyglutarate aldolase activity after electrophoresis of the enzyme on polyacrylamide gels. When gels are incubated with substrate (2-keto-4-hydroxyglutarate), activity is seen as a yellow-colored band due to interaction of the product )glyoxylate) with ortho-aminobenzaldehyde and glycine. Positive results have been obtained using either crude cell-free preparations or homogeneous enzyme from Escherichia coli as well as with highly purified samples of aldolase from bovine liver or kidney extracts. The method is potentially applicable to other aldolases that liberate an aliphatic aldehyde as a product; modifications and limitations of the procedure for detecting fructose 1,6-diphosphate aldolase, 2-keto-3-deoxy-6-phosphogluconate aldolase, and 2-deoxyribose-5-phosphate aldolase activities have been explored.     

2-Keto-4-hydroxyglutarate aldolase: purification and characterization of the homogeneous enzyme from bovine kidney.

2-Keto-4-hydroxyglutarate aldolase, which catalyzes the reversible cleavage of 2-keto-4-hydroxyglutarate, yielding pyruvate plus glyoxylate, has been purified from extracts of bovine kidney to apparent homogeneity as judged by polyacrylamide gel electrophoresis, gel filtration chromatography, sucrose density gradient centrifugation, and meniscus depletion sedimentation equilibrium experiments. The enzyme from this source has a native and a subunit mass of 144 and 36 kDa, respectively; the pH-activity optimum is 8.8. Rather than being stimulated, aldolase activity is inhibited to varying degrees by added divalent metal ions, whereas a number of metal ion-chelating agents have no effect. An absolute requirement for added thiol compounds could not be shown, but 2-mercaptoethanol enhances activity 2-fold, and added Hg2+ as well as p-mercuribenzoate or dithiodipyridine markedly inhibit catalysis. Incubation of the enzyme with either pyruvate or glyoxylate in the presence of NaBH4 causes extensive loss of aldolase activity concomitant with stable binding of approximately 1.0-1.5 mol of 14C-labeled substrate/mol of enzyme. The circular dichroism spectrum for native aldolase is characteristic of an alpha-helix; incubation of the enzyme with glyoxylate has no effect on this spectrum, but it is considerably altered by pyruvate. Bovine kidney aldolase shows no stereospecificity in catalyzing the aldol cleavage of the two optical isomers of 2-keto-4-hydroxyglutarate, and although it also catalyzes the beta-decarboxylation of oxalacetate, its decarboxylase/aldolase activity ratio is lower than that seen with the pure enzyme from either bovine liver or Escherichia coli.     

Identification of Escherichia coli YgaF as an L-2-hydroxyglutarate oxidase

YgaF, a protein of previously unknown function in Escherichia coli, was shown to possess noncovalently bound flavin adenine dinucleotide and to exhibit L-2-hydroxyglutarate oxidase activity. The inability of anaerobic, reduced enzyme to reverse the reaction by reducing the product alpha-ketoglutaric acid is explained by the very high reduction potential (+19 mV) of the bound cofactor. The likely role of this enzyme in the cell is to recover alpha-ketoglutarate mistakenly reduced by other enzymes or formed during growth on propionate. On the basis of the identified function, we propose that this gene be renamed lhgO.     

2-aminopurine allows interspecies recombination by a reversible inactivation of the Escherichia coli mismatch repair system

2-Aminopurine treatment of Escherichia coli induces a reversible phenotype of DNA mismatch repair deficiency. This transient phenotype results in a 300-fold increase in the frequency of interspecies conjugational recombination with a Salmonella enterica serovar Typhimurium Hfr donor. This method can be used for the generation of biodiversity by allowing recombination between diverged genes and genomes.     

Oxidation of 2-keto-4-hydroxyglutarate by pig heart and Escherichia coli alpha-ketoglutarate dehydrogenase complex.

Enzyme preparations from pig heart and Escherichia coli have been found to catalyze a NAD⁺- and CoASH-dependent oxidation of 2-keto-4-hydroxyglutarate. Several independent lines of evidence indicate that 2-keto-4-hydroxyglutarate is a substrate for the well-known α-ketoglutarate dehydrogenase complex of the citric acid cycle. The evidence includes (a) a constant ratio of specific activity values for the two substrates throughout purification, (b) identical elution profiles from a Ca₃(PO₄)₂ gel-cellulose column, (c) the same sucrose density sedimentation patterns, (d) similar responses in controlled heat inactivation studies, and (e) identical pH-activity curves.