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.     

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.     

Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase

Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA) displays optimal activity at 95 °C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthesis of 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde at the suboptimal temperature of 50 °C. The single mutant SacKdgA-V193A displayed a threefold increase in activity compared with wild type SacKdgA. The increased specific activity at 40–60 °C of this mutant was observed, not only for the condensation of pyruvate with glyceraldehyde, but also for several unnatural acceptor aldehydes. The optimal temperature for activity of SacKdgA-V193A was lower than for the wild type enzyme, but enzymatic stability of the mutant was similar to that of the wild type, indicating that activity and stability were uncoupled. Valine193 has Van der Waals interactions with Lysine153, which covalently binds the substrate during catalysis. The mutation V193A introduced space close to this essential residue, and the increased activity of the mutant presumably resulted from increased flexibility of Lysine153. The increased activity of SacKdgA-V193A with unaffected stability demonstrates the potential for optimizing extremely thermostable aldolases for synthesis reactions at moderate temperatures.     

Formation and cleavage of 2-keto-3-deoxygluconate by 2-keto-3-deoxygluconate aldolase of Aspergillus niger.

2-Keto-3-deoxygluconate aldolase of Aspergillus niger, an enzyme that has not been reported previously, was purified 468-fold. Maximal activity was obtained at pH 8.0 and 50 C. The enzyme exhibited relative stereochemical specificity with respect to glyceraldehyde. The Km values for 2-keto-3-deoxygluconate, glyceraldehyde, and pyruvate were 10, 13.3, and 3.0 mM, respectively. The effects of some compounds and inhibitors on enzyme activity were examined. Stability of the enzyme under different conditions was investigated. The equilibrium constant was about 0.33 X 10(-3) M.     

Specificity of 2-keto-3-deoxygluconate-6-P aldolase for open chain form of 2-keto-3-deoxygluconate-6-P.

2-Keto-3-deoxygluconate-6-P exists as an euqilibrium of three forms at 25 degrees measurable by 13C NMR: alpha-furanose anomer (41%), beta-furanose anomer (50%), and open chain keto (9%). The three forms are interconverted rapidly (greater than 0.5 s-1) so that the unidirectional rates of furanose ring opening and closing can be quantitated by an NMR line broadening method. The 2-keto-3-deoxygluconate aldolase is specific for only one of these forms, the open chain keto form. The rates for ring opening calculated from the rapid kinetic enzyme system compare closely with those obtained with the NMR method.     

Structure of 2-keto-3-deoxy-6-phosphogluconate aldolase at 2.8 Å resolution

The structure of 2-keto-3-deoxy-6-phosphogluconate aldolase has been extended to 2.8 Å resolution from 3.5 Å resolution by multiple isomorphous replacement methods using three heavy-atom derivatives and anomalous Bijvoet differences to 6 Å resolution (〈m〉 = 0.72). The replacement phases were improved and refined by electron density modification procedures coupled with inverse transform phase angle calculations. A Kendrew model of the molecule was built, which contained all 225 residues of a recently determined amino acid sequence, whereas only 173 were accounted for at 3.5 Å resolution. The missing residues were found to be part of the interior of the molecule and not simply an appendage. The molecule folds to form an eight-strand α/β-barrel structure strikingly similar to triosephosphate isomerase, the A-domain of pyruvate kinase and Taka amylase. With a knowledge of the sequence, the nature of the interfaces of the two kinds of crystallographic trimers have been examined, from which it was concluded that the choice of trimers selected in the 3.5 Å resolution work was probably correct for trimers in solution. The active site region has been established from the position of the Schiff base forming Lys144 but it has not been possible to confirm it conclusively in independent derivative experiments. An apparent anomaly exists in the location of Glu56 (about 25 Å from Lys144). The latter has been reported to assist in catalysis.     

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.     

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.     

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.     

On the interaction of 2-keto-3-deoxygalactonate-6P with 2-keto-3-deoxygluconate-6P aldolase ofPseudomonas putida. Evidence for enzyme-mediated,noncatalytic C-C bond turnover

2-Keto-3-deoxygluconate-6P (KDPG) aldolase ofPseudomonas putida mediates the cleavage of, as well as the condensation of, pyruvate andd-glyceraldehyde-3P (GaP) yielding, 2-keto-3-deoxygalactonate-6P (KDPGal) as side reactions of normal catalysis. These are visualized at high levels of aldolase. KDPGal cleavage occurs with aV _max that is 1/5000 that for KDPG cleavage. TheKm for KDPGal is 0.2 mM, with aK _i of 0.85 mM. The E-KDPGal complex is reductively inactivated having aKd of 0.55 mM. TheV/K value for KDPG cleavage is 2.0×10⁸ sec^?1, while the value for KDPGal cleavage is 1220 sec^?1. The difference in first-order rate constants of 164,000-fold argues that a step in the cleavage of KDPGal mediated by the enzyme is uncatalyzed. The enzyme is reductively inactivated by trapping the E-pyruvate, E-KDPG, or E-KDPGal complex. The enzyme can also be inactivated by reductive trapping of a catalytically nonproductive E-glyceraldehyde-3P complex. This latter occurs with aKd for GaP of 20 mM and a rate constant equivalent to a limiting half-time of 1110 sec at 1 mM cyanoborohydride. Reductive inactivation half-times in the presence of high GaP/KDPG ratios were the sum of both E-GaP and E-KDPG trapping by cyanoborohydride so that the inactivation rate due to KDPG could be determined. It was found at 1 mM cyanoborohydride that the limiting half-time for the E-KDPG complex was 2382 sec. The corresponding value for the E-KDPGal complex was 215 sec. Consequently, the E-KDPGal complex is 11 times more sensitive to reductive derivativation than is the E-KDPG complex. This is interpreted to show that the enzyme binds the KDPGal in a “normal” step forming a ketimine. However, turnover to the eneamine with resultant C-C bond cleavage is uncatalyzed. For the case of KDPGal synthesized by KDPG aldolase, it is argued that the pyruvate eneamine is bound to the active site, which can be attacked by GaP with its aldehyde carbon in the catalytically nonproductive conformation as a side reaction, presumably forming a tertiary complex. Spontaneous protonation of the resultant alcoholate anion would generate KDPGal. The data are interpreted to support an argument that catalytic proton turnover at the OH of C-4 of KDPG is required for normal catalysis, and that this step, which catalytically interconverts ketimine/eneamine, imposes steric constraints controlling the overall stereochemistry of the reaction.