Improving upon nature: active site remodeling produces highly efficient aldolase activity toward hydrophobic electrophilic substrates

The substrate specificity of enzymes is frequently narrow and constrained by multiple interactions, limiting the use of natural enzymes in biocatalytic applications. Aldolases have important synthetic applications, but the usefulness of these enzymes is hampered by their narrow reactivity profile with unnatural substrates. To explore the determinants of substrate selectivity and alter the specificity of Escherichia coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, we employed structure-based mutagenesis coupled with library screening of mutant enzymes localized to the bacterial periplasm. We identified two active site mutations (T161S and S184L) that work additively to enhance the substrate specificity of this aldolase to include catalysis of retro-aldol cleavage of (4S)-2-keto-4-hydroxy-4-(2'-pyridyl)butyrate (S-KHPB). These mutations improve the value of k(cat)/K(M)(S-KHPB) by >450-fold, resulting in a catalytic efficiency that is comparable to that of the wild-type enzyme with the natural substrate while retaining high stereoselectivity. Moreover, the value of k(cat)(S-KHPB) for this mutant enzyme, a parameter critical for biocatalytic applications, is 3-fold higher than the maximal value achieved by the natural aldolase with any substrate. This mutant also possesses high catalytic efficiency for the retro-aldol cleavage of the natural substrate, KDPG, and a >50-fold improved activity for cleavage of 2-keto-4-hydroxy-octonoate, a nonfunctionalized hydrophobic analogue. These data suggest a substrate binding mode that illuminates the origin of facial selectivity in aldol addition reactions catalyzed by KDPG and 2-keto-3-deoxy-6-phosphogalactonate aldolases. Furthermore, targeting mutations to the active site provides a marked improvement in substrate selectivity, demonstrating that structure-guided active site mutagenesis combined with selection techniques can efficiently identify proteins with characteristics that compare favorably to those of naturally occurring enzymes.     

Mutagenesis of the phosphate-binding pocket of KDPG aldolase enhances selectivity for hydrophobic substrates

Narrow substrate specificities often limit the use of enzymes in biocatalysis. To further the development of Escherichia coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase as a biocatalyst, the molecular determinants of substrate specificity were probed by mutagenesis. Our data demonstrate that S184 is located in the substrate-binding pocket and interacts with the phosphate moiety of KDPG, providing biochemical support for the binding model proposed on the basis of crystallographic data. An analysis of the substrate selectivity of the mutant enzymes indicates that alterations to the phosphate-binding site of KDPG aldolase changes the substrate selectivity. We report mutations that enhance catalysis of aldol cleavage of substrates lacking a phosphate moiety and demonstrate that electrophile reactivity correlates with the hydrophobicity of the substituted side chain. These mutations improve the selectivity for unnatural substrates as compared to KDPG by up to 2000-fold. Furthermore, the S184L KDPG aldolase mutant improves the catalytic efficiency for the synthesis of a precursor for nikkomycin by 40-fold, making it a useful biocatalyst for the preparation of fine chemicals.     

A bacterial selection for the directed evolution of pyruvate aldolases

A novel bacterial in vivo selection for pyruvate aldolase activity is described. Pyruvate kinase deficient cells, which lack the ability to biosynthetically generate pyruvate, require supplementation of exogenous pyruvate when grown on ribose. Supplementation with pyruvate concentrations as low as 50 microM rescues cell growth. A known substrate of the KDPG aldolases, 2-keto-4-hydroxy-4-(2'-pyridyl)butyrate (KHPB), also rescues cell growth, consistent with retroaldol cleavage by KDPG aldolase and rescue through pyruvate release. An initial round of selection against 2-keto-4-hydroxyoctonate (KHO), a nonsubstrate for wild-type aldolase, produced three mutants with intriguing alterations in protein sequence. This selection system allows rapid screening of mutant enzyme libraries and facilitates the discovery of enzymes with novel substrate specificities.     

Characterization and crystal structure of Escherichia coli KDPGal aldolase

2-Keto-3-deoxy-6-phosphogluconate (KDPG) and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases catalyze an identical reaction differing in substrate specificity in only the configuration of a single stereocenter. However, the proteins show little sequence homology at the amino acid level. Here we investigate the determinants of substrate selectivity of these enzymes. The Escherichia coli KDPGal aldolase gene, cloned into a T7 expression vector and overexpressed in E. coli, catalyzes retro-aldol cleavage of the natural substrate, KDPGal, with values of k(cat)/K(M) and k(cat) of 1.9x10(4)M(-1)s(-1) and 4s(-1), respectively. In the synthetic direction, KDPGal aldolase efficiently catalyzes an aldol addition using a limited number of aldehyde substrates, including d-glyceraldehyde-3-phosphate (natural substrate), d-glyceraldehyde, glycolaldehyde, and 2-pyridinecarboxaldehyde. A preparative scale reaction between 2-pyridinecarboxaldehyde and pyruvate catalyzed by KDPGal aldolase produced the aldol adduct of the R stereochemistry in >99.7% ee, a result complementary to that observed using the related KDPG aldolase. The native crystal structure has been solved to a resolution of 2.4A and displays the same (alpha/beta)(8) topology, as KDPG aldolase. We have also determined a 2.1A structure of a Schiff base complex between the enzyme and its substrate. This model predicts that a single amino acid change, T161 in KDPG aldolase to V154 in KDPGal aldolase, plays an important role in determining the stereochemical course of enzyme catalysis and this prediction was borne out by site-directed mutagenesis studies. However, additional changes in the enzyme sequence are required to prepare an enzyme with both high catalytic efficiency and altered stereochemistry.     

Mechanism of the Class I KDPG aldolase

In vivo, 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyzes the reversible, stereospecific retro-aldol cleavage of KDPG to pyruvate and D-glyceraldehyde-3-phosphate. The enzyme is a lysine-dependent (Class I) aldolase that functions through the intermediacy of a Schiff base. Here, we propose a mechanism for this enzyme based on crystallographic studies of wild-type and mutant aldolases. The three dimensional structure of KDPG aldolase from the thermophile Thermotoga maritima was determined to 1.9A. The structure is the standard alpha/beta barrel observed for all Class I aldolases. At the active site Lys we observe clear density for a pyruvate Schiff base. Density for a sulfate ion bound in a conserved cluster of residues close to the Schiff base is also observed. We have also determined the structure of a mutant of Escherichia coli KDPG aldolase in which the proposed general acid/base catalyst has been removed (E45N). One subunit of the trimer contains density suggesting a trapped pyruvate carbinolamine intermediate. All three subunits contain a phosphate ion bound in a location effectively identical to that of the sulfate ion bound in the T. maritima enzyme. The sulfate and phosphate ions experimentally locate the putative phosphate binding site of the aldolase and, together with the position of the bound pyruvate, facilitate construction of a model for the full-length KDPG substrate complex. The model requires only minimal positional adjustments of the experimentally determined covalent intermediate and bound anion to accommodate full-length substrate. The model identifies the key catalytic residues of the protein and suggests important roles for two observable water molecules. The first water molecule remains bound to the enzyme during the entire catalytic cycle, shuttling protons between the catalytic glutamate and the substrate. The second water molecule arises from dehydration of the carbinolamine and serves as the nucleophilic water during hydrolysis of the enzyme-product Schiff base. The second water molecule may also mediate the base-catalyzed enolization required to form the carbon nucleophile, again bridging to the catalytic glutamate. Many aspects of this mechanism are observed in other Class I aldolases and suggest a mechanistically and, perhaps, evolutionarily related family of aldolases distinct from the N-acetylneuraminate lyase (NAL) family.     

2-Keto-3-Deoxygluconate, an Intermediate in the Fermentation of Gluconate by Clostridia

Five clostridial species were found to ferment gluconate via 2-keto-3-deoxygluconate which subsequently is phosphorylated to yield 2-keto-3-deoxy-6-phosphogluconate (KDPG). This compound is then cleaved by KDPG aldolase.     

Cloning, isolation and characterization of the Thermotoga maritima KDPG aldolase

The Thermotoga maritima aldolase gene has been cloned into a T7 expression vector and overexpressed in Escherichia coli. The preparation yields 470 UL(-1) of enzyme at a specific activity of 9.4 U mg(-1). During retroaldol cleavage of KDPG, the enzyme shows a k(cat) that decreases with decreasing temperature. A more than offsetting decrease in K(m) yields an enzyme that is more efficient at 40 degrees C than at 70 degrees C. The substrate specificity of the enzyme was evaluated in the synthetic direction with a range of aldehyde substrates. Although the protein shows considerable structural homology to KDPG aldolases from mesophilic sources, significant differences in substrate specificity exist. A preparative scale reaction between 2-pyridine carboxaldehyde and pyruvate provided product of the same absolute configuration as mesophilic enzymes, but with diminished stereoselectivity.     

Pseudomonas cepacia mutants blocked in the Entner-Doudoroff pathway.

Pseudomonas cepacia mutants deficient in either 6-phosphogluconate (6PGA) dehydratase (Edd-) or 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (Eda-) failed to utilize glucose or gluconate despite the prominence of of 6-phosphogluconate dehydrogenase (6PGAD) ii this bacterium and the potential for utilizing the pentose shunt suggested by its growth on ribitol and xylose. The Eda- strains grew normally on glucuronic acid, indicating that in P. cepacia its degradation does not depend upon KDPG aldolase as it does in Escherichia coli. Both 6PGA dehydratase and KDPG aldolase were inducible enzymes, with 6PGA rather than gluconate the apparent inducer. Edd- as well as Eda- strains were sensitive to growth inhibition by glucose, gluconate, fructose, and related carbohydrates when these substrates were present in combination with alternate carbon sources such as citrate or phthalate, presumably as a consequence of accumulation and toxicity of 6PGA, KDPG, or both. Edd- mutants were somewhat less sensitive to such inhibition than were Eda- strains. Certain derivatives of the Edd- strains we examined were able to utilize gluconate despite their deficiency of 6PGA dehydratase. Such mutants formed higher levels of 6PGAD than did the wild type. It is likely that the elevated levels of 6PGAD in these strains prevents accumulation of toxic levels of 6PGA that would otherwise result from a block in he Entner-Doudoroff pathway. The results suggest that P. cepacia can mutate to grow slowly on gluconate utilizing only the pentose shunt.     

Purification of 2-keto-3-deoxy-6-phosphohexonate aldolases of Pseudomonas saccharophila

Summary KDPG aldolase has been crystallized from extracts of sucrosegrown cells of Pseudomonas saccharophila. KDPGal aldolase, which is absent in sucrose-grown cells but is present together with KDPG aldolase in galactose-grown cells has been purified over 1000-fold. The two enzymes share many common features, but possess absolute substrate specificity and are immunochemically distinct. The equilibrium constants for the catalyzed reactions have been determined and found to favor the synthesis of the 2-keto-3-deoxy-6-phosphohexonic acids.Dedicated to Prof. C. B. van Niel on the occasion of his 70th birthday. It was in his laboratory that Pseudomonas saccharophila was isolated and first studied some thirty years ago. In spite of a firm faith in enrichment culture techniques and incantations learned at Pacific Grove, repeated attempts to reisolate the organism from nature have been unsuccessful. One wonders whether it was the Knallgas atmosphere used for the original enrichment or that unique atmosphere of the master's laboratory, only occasionally tainted with a whiff of H₂S or mercury vapors, that nurtured this unique bacterium.This work was supported in part by a grant from the National Institutes of Health (AI-1808) and was conducted while the authors held tenure, respectively, as Postdoctoral Fellow and as Professor in the Miller Institute for Basic Research in Science at the University of California in Berkeley.     

Identification of biochemical and putative biological role of a xenolog from Escherichia coli using structural analysis

YagE is a 33 kDa prophage protein encoded by CP4-6 prophage element in Escherichia coli K12 genome. Here, we report the structures of YagE complexes with pyruvate (PDB Id 3N2X) and KDGal (2-keto-3-deoxy galactonate) (PDB Id 3NEV) at 2.2A resolution. Pyruvate depletion assay in presence of glyceraldehyde shows that YagE catalyses the aldol condensation of pyruvate and glyceraldehyde. Our results indicate that the biochemical function of YagE is that of a 2-keto-3-deoxy gluconate (KDG) aldolase. Interestingly, E. coli K12 genome lacks an intrinsic KDG aldolase. Moreover, the over-expression of YagE increases cell viability in the presence of certain bactericidal antibiotics, indicating a putative biological role of YagE as a prophage encoded virulence factor enabling the survival of bacteria in the presence of certain antibiotics. The analysis implies a possible mechanism of antibiotic resistance conferred by the over-expression of prophage encoded YagE to E. coli.     

Fermentation of pectin and glucose,and activity of pectin-degrading enzymes in the rabbit caecal bacterium Bifidobacterium pseudolongum

AIMS: In a rabbit caecal bacterium Bifidobacterium pseudolongum, metabolites of pectin and glucose, and activities of enzymes involved in the degradation of pectin were assayed. Simultaneously, activities of these enzymes were assayed in a rumen pectinolytic strain of Streptococcus bovis. METHODS AND RESULTS: A strain B. pseudolongum P6 which grew best on pectin was selected among bifidobacteria isolated from the rabbit caecum. Cultures of B. pseudolongum P6 grown on pectin produced significantly less formate, lactate and ethanol, and more acetate and succinate than cultures grown on glucose. No CO2 production on pectin was observed. Pectin macromolecule was degraded by extracellular pectinase (EC 3.2.1.15). Cell extracts possessed the activity of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EC 4.1.2.14). Streptococcus bovis X4, possessed activity of exopectate lyase and pectinase, but not that of KDPG aldolase. CONCLUSIONS: Our results are consistent with the assumption that in B. pseudolongum P6 acidic products of pectin degradation are catabolized via a modified Entner-Doudoroff pathway, as shown previously in rumen pectin-utilizing bacteria. The missing KDPG aldolase activity in Strep. bovis X4 seems to be the reason for the absence of growth of this bacterium on pectin. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on polysaccharide metabolism in bifidobacteria is fragmentary. This study extends the knowledge on pectin metabolism in intestinal bacteria.     

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.     

Crystal structure and functional assignment of YfaU, a metal ion dependent class II aldolase from Escherichia coli K12

One of the major challenges in the postgenomic era is the functional assignment of proteins using sequence- and structure-based predictive methods coupled with experimental validation. We have used these approaches to investigate the structure and function of the Escherichia coli K-12 protein YfaU, annotated as a putative 4-hydroxy-2-ketoheptane-1,7-dioate aldolase (HpcH) in the sequence databases. HpcH is the final enzyme in the degradation pathway of the aromatic compound homoprotocatechuate. We have determined the crystal structure of apo-YfaU and the Mg (2+)-pyruvate product complex. Despite greater sequence and structural similarity to HpcH, genomic context suggests YfaU is instead a 2-keto-3-deoxy sugar aldolase like the homologous 2-dehydro-3-deoxygalactarate aldolase (DDGA). Enzyme kinetic measurements show activity with the probable physiological substrate 2-keto-3-deoxy- l-rhamnonate, supporting the functional assignment, as well as the structurally similar 2-keto-3-deoxy- l-mannonate and 2-keto-3-deoxy- l-lyxonate (see accompanying paper: Rakus, J. F., Fedorov, A. A., Fedorov, E. V., Glasner, M. E., Hubbard, B. K., Delli, J. D., Babbitt, P. C., Almo, S. C., and Gerlt, J. A. (2008) Biochemistry 47, 9944-9954). YfaU has similar activity toward the HpcH substrate 4-hydroxy-2-ketoheptane-1,7-dioate and synthetic substrates 4-hydroxy-2-ketopentanoic acid and 4-hydroxy-2-ketohexanoic acid. This indicates a relaxed substrate specificity that complicates the functional assignment of members of this enzyme superfamily. Crystal structures suggest these enzymes use an Asp-His intersubunit dyad to activate a metal-bound water or hydroxide for proton transfer during catalysis.     

Crystal structure and stereochemical studies of KD(P)G aldolase from Thermoproteus tenax

Carbon-carbon bond forming enzymes offer great potential for organic biosynthesis. Hence there is an ongoing effort to improve their biocatalytic properties, regarding availability, activity, stability, and substrate specificity and selectivity. Aldolases belong to the class of C-C bond forming enzymes and play important roles in numerous cellular processes. In several hyperthermophilic Archaea the 2-keto-3-deoxy-(6-phospho)-gluconate (KD(P)G) aldolase was identified as a key player in the metabolic pathway. The carbohydrate metabolism of the hyperthermophilic Crenarchaeote Thermoproteus tenax, for example, has been found to employ a combination of a variant of the Embden-Meyerhof-Parnas pathway and an unusual branched Entner-Doudoroff pathway that harbors a nonphosphorylative and a semiphosphorylative branch. The KD(P)G aldolase catalyzes the reversible cleavage of 2-keto-3-deoxy-6-phosphogluconate (KDPG) and 2-keto-3-deoxygluconate (KDG) forming pyruvate and glyceraldehyde 3-phosphate or glyceraldehyde, respectively. In T. tenax initial studies revealed that the pathway is specific for glucose, whereas in the thermoacidophilic Crenarchaeote Sulfolobus solfataricus the pathway was shown to be promiscuous for glucose and galactose degradation. The KD(P)G aldolase of S. solfataricus lacks stereo control and displays additional activity with 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) and 2-keto-3-deoxygalactonate (KDGal), similar to the KD(P)G aldolase of Sulfolobus acidocaldarius. To address the stereo control of the T. tenax enzyme the formation of the two C4 epimers KDG and KDGal was analyzed via gas chromatography combined with mass spectroscopy. Furthermore, the crystal structure of the apoprotein was determined to a resolution of 2.0 A, and the crystal structure of the protein covalently linked to a pathway intermediate, namely pyruvate, was determined to 2.2 A. Interestingly, although the pathway seems to be specific for glucose in T. tenax the enzyme apparently also lacks stereo control, suggesting that the enzyme is a trade-off between required catabolic flexibility needed for the conversion of phosphorylated and nonphosphorylated substrates and required stereo control of cellular/physiological enzymatic reactions.     

Fermentation of pectin and glucose, and activity of pectin-degrading enzymes in the rabbit caecal bacterium Bacteroides caccae

AIMS: To compare fermentation pattern in cultures of Bacteroides caccae supplied with pectin and glucose, and identify enzymes involved in metabolism of pectin. METHODS AND RESULTS: A strain KWN isolated from the rabbit caecum was used. Fermentation pattern, changes of viscosity and enzyme reactions products were determined. Cultures grown on pectin produced significantly more acetate and less formate, lactate, fumarate and succinate than cultures grown on glucose. Production of cell dry matter and protein per gram of substrate used was the same in pectin- and glucose-grown cultures. The principal enzymes that participated in the metabolism of pectin were extracellular exopectate hydrolase (EC 3.2.1.67), extracellular endopectate lyase (EC 4.2.2.2) and cell-associated 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EC 4.1.2.14). The latter enzyme is unique to the Entner-Doudoroff pathway. Activities of pectinolytic enzymes in cultures grown on glucose were low. Activity of KDPG aldolase was similar in pectin- and glucose-grown cells. CONCLUSIONS: Metabolites and activities of pectin-degrading enzymes differed in cultures of B. caccae KWN grown on pectin and glucose. Yields of dry matter and protein were the same on both substrates. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on metabolism of pectin in animal strains of Bacteroides is incomplete. This study extends the knowledge on metabolism in bacteria from the rabbit caecum.     

利用EDA融合标签高效表达猪圆环病毒2型壳蛋白

原核生物作为宿主细胞被广泛应用于异源蛋白质的重组表达,并且为生物活性蛋白质的制备提供了一种高效、经济的方法,因而在分子生物学中得到普遍的应用。然而,病毒蛋白在使用原核重组表达系统进行重组表达时,会出现病毒蛋白溶解性差和表达量低等问题。因此,通过使用各种融合标签以增加目的重组蛋白的表达量和溶解性成为有效的方法。本研究通过使用3种融合标签（EDA标签、MBP标签和GST标签）以获得表达量高的可溶性重组表达猪圆环病毒2型壳蛋白;并比较3种融合标签对该蛋白表达量、溶解性和稳定性的影响。研究结果表明,EDA标签可以显著提高重组表达的猪圆环病毒2型壳蛋白表达量,并且能够增强该蛋白的稳定性;MBP标签可增强重组表达的猪圆环病毒2型壳蛋白表达量,但是不能改善该蛋白的稳定性;GST标签能够增强该重组表达蛋白的表达量,但是不能增强该蛋白的溶解性和稳定性。本研究将EDA作为PCV2-CP蛋白的融合标签,显著提高PCV2-CP-EDA重组蛋白的表达量和增强该重组蛋白的稳定性,为病毒蛋白的可溶性重组表达提供了一种新的融合标签。     

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.     

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.     

The Nonphosphorylative Entner-Doudoroff Pathway in the Thermoacidophilic Euryarchaeon Picrophilus torridus Involves a Novel 2-Keto-3-Deoxygluconate- Specific Aldolase

The pathway of glucose degradation in the thermoacidophilic euryarchaeon Picrophilus torridus has been studied by in vivo labeling experiments and enzyme analyses. After growth of P. torridus in the presence of [1-¹³C]- and [3-¹³C]glucose, the label was found only in the C-1 and C-3 positions, respectively, of the proteinogenic amino acid alanine, indicating the exclusive operation of an Entner-Doudoroff (ED)-type pathway in vivo. Cell extracts of P. torridus contained all enzyme activities of a nonphosphorylative ED pathway, which were not induced by glucose. Two key enzymes, gluconate dehydratase (GAD) and a novel 2-keto-3-deoxygluconate (KDG)-specific aldolase (KDGA), were characterized. GAD is a homooctamer of 44-kDa subunits, encoded by Pto0485. KDG aldolase, KDGA, is a homotetramer of 32-kDa subunits. This enzyme was highly specific for KDG with up to 2,000-fold-higher catalytic efficiency compared to 2-keto-3-deoxy-6-phosphogluconate (KDPG) and thus differs from the bifunctional KDG/KDPG aldolase, KD(P)GA of crenarchaea catalyzing the conversion of both KDG and KDPG with a preference for KDPG. The KDGA-encoding gene, kdgA, was identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) as Pto1279, and the correct translation start codon, an ATG 24 bp upstream of the annotated start codon of Pto1279, was determined by N-terminal amino acid analysis. The kdgA gene was functionally overexpressed in Escherichia coli. Phylogenetic analysis revealed that KDGA is only distantly related to KD(P)GA, both enzymes forming separate families within the dihydrodipicolinate synthase superfamily. From the data we conclude that P. torridus degrades glucose via a strictly nonphosphorylative ED pathway with a novel KDG-specific aldolase, thus excluding the operation of the branched ED pathway involving a bifunctional KD(P)GA as a key enzyme.Comparative analyses of sugar-degrading pathways in members of the domain Archaea revealed that all species analyzed so far degrade glucose and glucose polymers to pyruvate via modification of the classical Embden-Meyerhof (EM) and Entner-Doudoroff (ED) pathways found in bacteria and eukarya. Modified EM pathways were reported for hyperthermophilic archaea, including, e.g., the strictly fermentative Thermococcales and Desulfurococcales, the sulfur-reducing Thermoproteus tenax, and the microaerophilic Pyrobaculum aerophilum. These pathways differ from the classical EM pathway by the presence of several novel enzymes and enzyme families, catalyzing, e.g., the phosphorylation of glucose and fructose-6-phosphate, isomerization of glucose-6-phosphate, and oxidation of glyceraldehyde-3-phosphate (18, 22, 25).Modified ED pathways have been proposed for aerobic archaea, including halophiles, and thermoacidophilic crenarchaea, such as Sulfolobus species, and the euryarchaea Thermoplasma acidophilum and Picrophilus torridus. The anaerobic Thermoproteus tenax, which degrades glucose predominantly via a modified EM pathway, also utilizes—to a minor extent (<20%)—a modified ED pathway for glucose degradation. The following ED pathway modifications have been reported in archaea (25). A semiphosphorylative ED pathway was reported in halophilic archaea. Accordingly, glucose is converted to 2-keto-3-deoxy-6-gluconate (KDG) via glucose dehydrogenase and gluconate dehydratase. KDG is then phosphorylated by KDG kinase to 2-keto-3-deoxy-6-phosphogluconate (KDPG), which is split by KDPG aldolase to pyruvate and glyceraldehyde-3-phosphate (GAP). GAP is further converted to form another pyruvate via common reactions of the EM pathway, i.e., phosphorylative GAP dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase. The net ATP yield of this pathway is 1 ATP/mol glucose.From initial enzyme studies of the thermoacidophilic archaea Sulfolobus solfataricus, Thermoplasma acidophilum, and Thermoproteus tenax, a nonphosphorylative ED pathway was proposed (25). In this modification of the ED pathway, glucose is converted to KDG via glucose dehydrogenase and gluconate dehydratase, as in the semiphosphorylative pathway, but then the steps differ as follows: KDG is cleaved into pyruvate and glyceraldehyde via 2-keto-3-deoxygluconate-specific aldolase (KDGA). The subsequent oxidation of glyceraldehyde to glycerate involves either NAD(P)⁺-dependent dehydrogenases or oxidoreductases. Glycerate is then phosphorylated by a specific kinase to 2-phosphoglycerate, which is finally converted to pyruvate via enolase and pyruvate kinase. This modification of the ED pathway was called “nonphosphorylative” since it is not coupled with net ATP synthesis.However, recent comparative genomic studies and refined enzyme analyses suggest that the crenarchaea Sulfolobus and Thermoproteus utilize a so-called branched ED pathway, in which a semiphosphorylated route is simultaneously operative in addition to the nonphosphorylative route (25, 32). Accordingly, the semiphosphorylated route involves—via KDG kinase—the phosphorylation of KDG to KDPG, which is then cleaved to pyruvate and GAP by means of a bifunctional KDG/KDPG aldolase, KD(P)GA. GAP is then converted to another pyruvate via nonphosphorylative GAP dehydrogenase (GAPN), phosphoglycerate mutase, enolase, and pyruvate kinase. The net ATP yield of the branched ED pathway is zero. In support of this pathway, the genes encoding gluconate dehydratase, bifunctional KD(P)GA, KDG kinase, and GAPN were found to be clustered in Sulfolobus solfataricus (see Discussion) and Thermoproteus tenax. The key enzyme of the proposed branched ED pathway is the bifunctional KD(P)GA, which catalyzes the cleavage of KDG to pyruvate and glyceraldehyde and cleavage of KDPG to pyruvate and glyceraldehyde-3-phosphate. This bifunctional aldolase, which has been characterized from S. solfataricus, was found to be identical to a previously described KDG aldolase of the same organism; however, its catalytic property to also utilize KDPG as a substrate has been recognized only recently. In fact, the bifunctional KD(P)GA showed a higher catalytic efficiency for KDPG than for KDG (1, 14). Crystal structures of bifunctional KD(P)GAs of S. solfataricus and T. tenax have been reported (16, 27, 30; G. Taylor [United Kingdom], unpublished data).The branched ED pathway in S. solfataricus has been reported to be promiscuous and therefore represents an equivalent degradation route for both glucose and its C-4 epimer, galactose. Accordingly, glucose dehydrogenase, gluconate dehydratase, KDG kinase, and bifunctional KD(P)GA were found to catalyze the conversion of both glucose and galactose and the corresponding subsequent intermediates, i.e., gluconate/galactonate, KDG/KDGal (KDGal stands for 2-keto-3-deoxygalactonate), and KDPG/KDPGal (KDPGal stands for 2-keto-3-deoxy-6-phosphogalactonate) (4, 12-14).In contrast to crenarchaea, the modified ED pathway in the thermoacidophilic euryarchaea Thermoplasma acidophilum and Picrophilus torridus has not been studied in detail. Enzyme measurements in cell extracts and the characterization of few enzymes suggest the operation of a nonphosphorylative ED pathway in these organisms (2, 3, 17, 19, 25). However, in vivo evidence for the operation of an ED-type pathway, e.g., by ¹³C-labeling experiments with growing cultures, has not been provided yet. Furthermore, the KDG aldolase activity measured in cell extracts of P. torridus and T. acidophilum has not been purified and characterized, in particular with respect to substrate specificity, and the genes encoding these enzymes have not been identified. The biochemical analysis of this aldolase is crucial to define the enzyme as a KDG-specific aldolase, indicative of a nonphosphorylative ED pathway, or as bifunctional KD(P)GA, indicative of the branched ED pathway as proposed for the crenarchaea Sulfolobus and Thermoproteus.In this communication we studied the sugar-degrading pathway in P. torridus by in vivo labeling experiments with [¹³C]glucose, by enzyme measurements, and by characterization of two key enzymes, gluconate dehydratase and KDG aldolase. The data indicate that P. torridus utilizes a strict nonphosphorylative ED pathway, involving a novel KDG-specific aldolase as a key enzyme, and thus exclude the operation of a branched ED pathway, as in crenarchaea involving a bifunctional KD(P)GA as a key enzyme.     

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.