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.     

Production of 2-keto-3-deoxygluconate by immobilized cells of Sulfolobus solfataricus

Summary 2-Keto-3-deoxygluconate, an intermediate of glucose breakdown inSulfolobus solfataricus, was produced by enzymic dehydration of gluconate using whole cells of the micro-organism immobilized in crude egg white. The degradation of 2-keto-3-deoxygluconate to pyruvate and glyceraldehyde was avoided by inhibiting the aldolase activity in the cells by sodium borohydride treatment.     

Metabolic pathway promiscuity in the archaeon Sulfolobus solfataricus revealed by studies on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase

The hyperthermophilic Archaeon Sulfolobus solfataricus metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate (KDG) aldolase then catalyzes the cleavage of 2-keto-3-deoxygluconate to glyceraldehyde and pyruvate. The gene encoding glucose dehydrogenase has been cloned and expressed in Escherichia coli to give a fully active enzyme, with properties indistinguishable from the enzyme purified from S. solfataricus cells. Kinetic analysis revealed the enzyme to have a high catalytic efficiency for both glucose and galactose. KDG aldolase from S. solfataricus has previously been cloned and expressed in E. coli. In the current work its stereoselectivity was investigated by aldol condensation reactions between D-glyceraldehyde and pyruvate; this revealed the enzyme to have an unexpected lack of facial selectivity, yielding approximately equal quantities of 2-keto-3-deoxygluconate and 2-keto-3-deoxygalactonate. The KDG aldolase-catalyzed cleavage reaction was also investigated, and a comparable catalytic efficiency was observed with both compounds. Our evidence suggests that the same enzymes are responsible for the catabolism of both glucose and galactose in this Archaeon. The physiological and evolutionary implications of this observation are discussed in terms of catalytic and metabolic promiscuity.     

2-keto-3-deoxygluconate transport system in Erwinia chrysanthemi.

In Erwinia chrysanthemi, the gene kdgT encodes a transport system responsible for the uptake of ketodeoxyuronates. We studied the biochemical properties of this transport system. The bacteria could grow on 2,5-diketo-3-deoxygluconate but not on 2-keto-3-deoxygluconate. The 2-keto-3-deoxygluconate entry reaction displayed saturation kinetics, with an apparent Km of 0.52 mM (at 30 degrees C and pH 7). 5-Keto-4-deoxyuronate and 2,5-diketo-3-deoxygluconate appeared to be competitive inhibitors, with Kis of 0.11 and 0.06 mM, respectively. The 2-keto-3-deoxygluconate permease could mediate the uptake of glucuronate with a low affinity. kdgT was cloned on an R-prime plasmid formed by in vivo complementation of a kdgT mutation of Escherichia coli. After being subcloned, it was mutagenized with a mini-Mu-lac transposable element able to form fusions with the lacZ gene. We introduced a kdgT-lac fusion into the E. chrysanthemi chromosome by marker exchange recombination and studied its regulation. kdgT product synthesis was not induced by external 2-keto-3-deoxygluconate in the wild-type strain but was induced by galacturonate and polygalacturonate. Two types of regulatory mutants able to grow on 2-keto-3-deoxygluconate as the sole carbon source were studied. Mutants of one group had a mutation in the operator region of kdgT; mutants of the other group had a mutation in kdgR, a regulatory gene controlling kdgT expression.     

Gene expression and characterization of 2-keto-3-deoxygluconate kinase, a key enzyme in the modified Entner-Doudoroff pathway of the aerobic and acidophilic hyperthermophile Sulfolobus tokodaii

2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the ATP-dependent phosphorylation of 2-keto-3-deoxygluconate, a key intermediate in the modified (semi-phosphorylative) Entner-Doudoroff (ED) glucose metabolic pathway. We identified the gene (ORF ID: ST2478) encoding KDGK in the hyperthermophilic archaeon Sulfolobus tokodaii based on the structure of a gene cluster in a genomic database and functionally expressed it in Escherichia coli. The expressed protein was purified from crude extract by heat treatment and two conventional column chromatography steps, and the partial amino acid sequence in the N-terminal region of the purified enzyme (MAKLIT) was identical to that obtained from the gene sequence. The purified enzyme was extremely thermostable and retained full activity after heating at 80 degrees C for 1 h. The enzyme utilized ATP or GTP, but not ADP or AMP, as a phosphoryl donor and 2-keto-3-deoxy-D-gluconate or 2-keto-D-gluconate as a phosphoryl acceptor. Divalent cations including Mg(2+), Co(2+), Ni(2+), Zn(2+) or Mn(2+) were required for activity, and the apparent Km values for KDG and ATP at 50 degrees C were 0.027 mM and 0.057 mM, respectively. The presence of KDGK means that the hyperthermophilic archaeon S. tokodaii metabolizes glucose via both modified (semi-phosphorylative) and non-phosphorylative ED pathways.     

The structural basis for substrate promiscuity in 2-keto-3-deoxygluconate aldolase from the Entner-Doudoroff pathway in Sulfolobus solfataricus

The hyperthermophilic Archaea Sulfolobus solfataricus grows optimally above 80 degrees C and metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to D-2-keto-3-deoxygluconate (KDG). KDG aldolase (KDGA) then catalyzes the cleavage of KDG to D-glyceraldehyde and pyruvate. It has recently been shown that all the enzymes of this pathway exhibit a catalytic promiscuity that also enables them to be used for the metabolism of galactose. This phenomenon, known as metabolic pathway promiscuity, depends crucially on the ability of KDGA to cleave KDG and D-2-keto-3-deoxygalactonate (KDGal), in both cases producing pyruvate and D-glyceraldehyde. In turn, the aldolase exhibits a remarkable lack of stereoselectivity in the condensation reaction of pyruvate and D-glyceraldehyde, forming a mixture of KDG and KDGal. We now report the structure of KDGA, determined by multiwavelength anomalous diffraction phasing, and confirm that it is a member of the tetrameric N-acetylneuraminate lyase superfamily of Schiff base-forming aldolases. Furthermore, by soaking crystals of the aldolase at more than 80 degrees C below its temperature activity optimum, we have been able to trap Schiff base complexes of the natural substrates pyruvate, KDG, KDGal, and pyruvate plus D-glyceraldehyde, which have allowed rationalization of the structural basis of promiscuous substrate recognition and catalysis. It is proposed that the active site of the enzyme is rigid to keep its thermostability but incorporates extra functionality to be promiscuous.     

Structure of Thermus thermophilus 2-Keto-3-deoxygluconate kinase: evidence for recognition of an open chain substrate

2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the phosphorylation of 2-keto-3-deoxygluconate (KDG) to 2-keto-3-deoxy-6-phosphogluconate (KDGP). The genome sequence of Thermus thermophilus HB8 contains an open reading frame that has a 30% identity to Escherichia coli KDGK. The KDGK activity of T.thermophilus protein (TtKDGK) has been confirmed, and its crystal structure has been determined by the molecular replacement method and refined with two crystal forms to 2.3 angstroms and 3.2 angstroms, respectively. The enzyme is a hexamer organized as a trimer of dimers. Each subunit is composed of two domains, a larger alpha/beta domain and a smaller beta-sheet domain, similar to that of ribokinase and adenosine kinase, members of the PfkB family of carbohydrate kinases. Furthermore, the TtKDGK structure with its KDG and ATP analogue was determined and refined at 2.1 angstroms. The bound KDG was observed predominantly as an open chain structure. The positioning of ligands and the conservation of important catalytic residues suggest that the reaction mechanism is likely to be similar to that of other members of the PfkB family, including ribokinase. In particular, the Asp251 is postulated to have a role in transferring the gamma-phosphate of ATP to the 5'-hydroxyl group of KDG.     

Nucleotide sequence of the Erwinia chrysanthemi gene encoding 2-keto-3-deoxygluconate permease

The phytopathogenic bacterium Erwinia chrysanthemi produces a group of pectolytic enzymes able to depolymerise the pectic compounds in plant cell walls. The resulting tissue maceration is known as soft rot disease. The degraded pectin products are transported by 2-keto-3-deoxygluconate permease into the bacterial cell, where they serve as carbon and energy sources. This H+ coupled transport system is encoded by the kdgT gene; we report the nucleotide sequence of kdgT. It is encoded by an open reading frame (ORF) of 1194 bp, which is preceded by an Escherichia coli-type promoter region. The ORF encodes a protein with 398 amino acid (aa) residues and a predicted Mr of 48,550. As would be expected for a membrane protein, it is very hydrophobic, containing 63% nonpolar aa. However, the kdgT gene has no apparent evolutionary relationship to other genes encoding sugar transport proteins, such as lacY, melB or the E. coli citrate transport gene. Southern hybridization experiments indicate a strong homology between the Er. chrysanthemi and E. coli kdgT genes; there is also a second region on the E. coli chromosome with homology to kdgT. The kdgT gene is located near the ade-377 marker on the Er. chrysanthemi chromosome (equivalent to the region between 20 and 30 min in E. coli), whereas the E. coli kdgT gene is located at 88 min. Thus, these two enterobacteria show some significant differences in their genomic organization.     

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.     

Identification of 3-deoxyglucosone dehydrogenase as aldehyde dehydrogenase 1A1 (retinaldehyde dehydrogenase 1)

One of the metabolic fates of 3-deoxyglucosone, a product of protein deglycation and a potent glycating agent, is to be oxidized to 2-keto-3-deoxygluconate, but the enzyme that catalyzes this reaction is presently unknown. Starting from human erythrocytes, which are known to convert 3-deoxyglucosone to 2-keto-3-deoxygluconate, we have purified to near homogeneity a NAD-dependent dehydrogenase that catalyzes this last reaction at neutral pH. Sequencing of a 55 kDa band co-eluting with the enzymatic activity in the last step indicated that it corresponded to aldehyde dehydrogenase 1A1 (ALDH1A1), an enzyme known to catalyze the oxidation of retinaldehyde to retinoic acid. Overexpression of human ALDH1A1 in HEK cells led to a more than 20-fold increase in 3-deoxyglucosone dehydrogenase activity. In mouse tissues 3-deoxyglucosone dehydrogenase activity was highest in liver, intermediate in lung and testis, and negligible or undetectable in other tissues, in agreement with the tissue distribution of ALDH1A1 mRNA. 3-deoxyglucosone dehydrogenase activity was undetectable in tissues from ALDH1A1(-/-) mice. ALDH1A1 appears therefore to be the major if not the only enzyme responsible for the oxidation of 3-deoxyglucosone to 2-keto-3-deoxygluconate. The urinary excretion of 2-keto-3-deoxygluconate amounted to 16.7 micromol/g creatinine in humans, indicating that 3-deoxyglucosone may be quantitatively a more important substrate than retinaldehyde for ALDH1A1.     

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.     

Glyceraldehyde dehydrogenases from the thermoacidophilic euryarchaeota Picrophilus torridus and Thermoplasma acidophilum, key enzymes of the non-phosphorylative Entner-Doudoroff pathway, constitute a novel enzyme family within the aldehyde dehydrogenase superfamily

Cells of Picrophilus torridus, grown on glucose, contained all enzyme activities of a non-phosphorylative Entner-Doudoroff pathway, including glucose dehydrogenase, gluconate dehydratase, 2-keto-3-deoxygluconate aldolase, glyceraldehyde dehydrogenase (GADH), glycerate kinase (2-phosphoglycerate forming), enolase and pyruvate kinase. GADH was purified to homogeneity. The 115-kDa homodimeric protein catalyzed the oxidation of glyceraldehyde with NADP+ at highest catalytic efficiency. NAD+ was not used. By MALDI-TOF analysis, open reading frame (ORF) Pto0332 was identified in the genome of P. torridus as the encoding gene, designated gadh, and the recombinant GADH was characterized. In Thermoplasma acidophilum ORF Ta0809 represents a gadh homolog with highest sequence identity; the gene was expressed and the recombinant protein was characterized as functional GADH with properties very similar to the P. torridus enzyme. Sequence comparison and phylogenetic analysis define both GADHs as members of novel enzyme family within the aldehyde dehydrogenase superfamily.     

Biochemical and structural exploration of the catalytic capacity of Sulfolobus KDG aldolases

Aldolases are enzymes with potential applications in biosynthesis, depending on their activity, specificity and stability. In the present study, the genomes of Sulfolobus species were screened for aldolases. Two new KDGA [2-keto-3-deoxygluconate (2-oxo-3-deoxygluconate) aldolases] from Sulfolobus acidocaldarius and Sulfolobus tokodaii were identified, overexpressed in Escherichia coli and characterized. Both enzymes were found to have biochemical properties similar to the previously characterized S. solfataricus KDGA, including the condensation of pyruvate and either D,L-glyceraldehyde or D,L-glyceraldehyde 3-phosphate. The crystal structure of S. acidocaldarius KDGA revealed the presence of a novel phosphate-binding motif that allows the formation of multiple hydrogen-bonding interactions with the acceptor substrate, and enables high activity with glyceraldehyde 3-phosphate. Activity analyses with unnatural substrates revealed that these three KDGAs readily accept aldehydes with two to four carbon atoms, and that even aldoses with five carbon atoms are accepted to some extent. Water-mediated interactions permit binding of substrates in multiple conformations in the spacious hydrophilic binding site, and correlate with the observed broad substrate specificity.     

Immobilized flounder muscle glyceraldehyde 3-phosphate dehydrogenase

Summary Partially purified flounder muscle (Pseudopleuronectus americanus) glyceraldehyde 3-phosphate dehydrogenase was immobilized on cyanogen bromide-activated Sepharose. The catalytic properties of the immobilized preparation were studied to determine if immobilization alters the kinetic properties of the native holoenzyme. The results indicate that the pH activity profile of immobilized glyceraldehyde 3-phosphate dehydrogenase did not differ from that of the native enzyme. The Michaelis constants (Km) for NAD and glyceraldehyde 3-phosphate were somewhat altered. The enzyme stability toward various inactivation treatments in the presence and absence of NAD was characterized and compared to that of he native enzyme. When either form of the enzyme was incubated with urea at concentrations greater than 2m, inactivation occurred very rapidly. Incubation in 0.1% trypsin for 60 minutes decreased the activity of immobilized glyceraldehyde 3-phosphate dehydrogenase by 45% and of the native soluble enzyme by 70%. The immobilized enzyme also exhibited considerably more stability than the native soluble enzyme when exposed to a temperature of 50° or to 20 mm ATP. In all cases NAD either greatly reduced the rate of inactivation or completely protected the enzyme from inactivation.     

The development of SS''-polymethylenebis(methanethiosulphonates) as reversible cross-linking reagents for thiol groups and their use to form stable catalytically active cross-linked dimers within glyceraldehyde 3-phosphate dehydrogenase.

The synthesis of a series of SS'-polymethylenebis(methanethiosulphonates) including the pentane, hexane, octane, decane and dodecane derivatives is described. These derivatives were synthesized by condensation between dibromoalkanes and potassium methanethiosulphonate in refluxing methanol and this seems an especially versatile reaction for the synthesis of asymmetric thiosulphonate derivatives. The synthesis of SS'-[1,8-3H4]-octamethylenebis(methanethiosulphonate) was also perfomed. Cross-linking was demonstrated in the four enzymes lactate dehydrogenase, phosphofructokinase, pyruvate kinase and glyceraldehyde 3-phosphate dehydrogenase. For all four enzymes cross-linking was efficiently reversed by reducing conditions in denaturing solvents. The reaction with glyceraldehyde 3-phosphate dehydrogenase was unique in that only the cross-linked dimer was produced in significant amounts (greater than 90% of total products as dimer). This reaction was followed in detail with radioactive cross-linking reagent. Inhibition of enzyme activity was extremely fast and showed an asymmetric distribution of enzyme activity on subunits. Thus complete modification of only one subunit resulted in up to 75% inhibition of enzyme activity. Reaction of glyceraldehyde 3-phosphate dehydrogenase with 1.25 mol of SS'-octamethylenebis(methanethiosulphonate) per mol of enzyme subunit produced two species of protein. The first species was obtained in 20% yield and was only partially re-activated on mild reduction with 2-mercaptoethanol. The second species was isolated in 66% yield and was completely re-activated on mild reduction. Before reduction there was 4 mol of inhibitor per tetramer for the latter species, and more than 95% of the enzyme was present as a dimer on non-reducing electrophoresis. After mild reduction 2 mol of inhibitor was still bound per tetramer, the enzyme was now catalytically active and the dimer was still the major structure on non-reducing electrophoresis. Thus mild reduction of SS'-octamethylenebis(methanethiosulphonate-treated glyceraldehyde 3-phosphate dehydrogenase enabled the production of active enzyme in which there is a stable cross-link across one of the molecular axes of the tetrameric enzyme. This cross-link was only reversed if reduction was performed when the enzyme was denatured. The molecular weight of cross-linked and re-activated cross-linked glyceraldehyde 3-phosphate dehydrogenase was established as 144000 (tetramer) by sucrose-density-gradient centrifugation. These observations are interpreted in terms of the molecular structure of glyceraldehyde 3-phosphate dehydrogenase.     

Modification of glyceraldehyde 3-phosphate dehydrogenase activity by adsorption on phospholipid vesicles.

1. The adsorption of [14C]carboxymethylated glyceraldehyde 3-phosphate dehydrogenase to negatively charged liposomes of phsphatidic acid/phosphatidylcholine (3:7, w/w) was investigated. The apparent association constant at I/2 = 60, pH 7.6, was 0.4 X 10(6)M-1. Adsorption decreased as ionic strength and pH were increased. 2. In the presence of negatively charged liposomes, the Km value for glyceraldehyde 3-phosphate of glyceraldehyde 3-phosphate dehydrogenase was increased and Vmax. decreased. In the presence of positively charged liposomes, the Km value for glyceraldehyde 3-phosphate decreased and there was no significant change in Vmax. Addition of Triton X-100 abolished the effect of both positively and negatively charged liposomes on the kinetic properties of the enzyme.     

Enzymatic synthesis of 2-keto-3-deoxy-d-glucose from D-gluconate

2-Keto-3-deoxy-d-gluconate (KDG¹) was prepared from d-gluconate with a cell-free extract of Clostridium pasteurianum containing gluconate dehydratase. This enzyme is present in gluconate-grown cells in high activity. Because of the heat tolerance of the dehydratase KDG synthesis could be carried out at 50°C. Sodium-KDG was isolated from the reaction mixture as crude product with a yield of 96%. The crystalline free acid was prepared from the sodium salt in 40% yield.     

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.     

Glyceraldehyde 3-phosphate dehydrogenase of Scenedesmus obliquus: Promotion of NADPH-dependent activity and antagonisation by NAD

The glyceraldehyde 3-phosphate dehydrogenase activity of extracts from heterotrophic Scenedesmus obliquus was linked predominantly to NADH. However, on DEAE-cellulose chromatography the enzyme was eluted by a gradient of phosphate in a form characterized by high NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase activity. This interconversion of enzyme forms could be prevented by the presence of NAD during DEAE-cellulose chromatography.High concentrations of phosphate stimulated the NADPH-dependent activity of the purified enzyme at the expense of activity linked to NADH and these changes were associated with depolymerization of a hexadecamer to a tetramer. The effect of phosphate on the rates of increase in NADPH-dependent activity and of a decrease in activity linked to NADH was cooperative with a Hill coefficient of 3.2. The inversely related changes in coenzyme specificity were inhibited to the same extent by NAD and the response to this ligand was anticooperative. These findings imply a strictly inverse proportional relationship between the rates of change of NADH and NADPH-linked activity. In the presence of dithiothreitol, low concentrations of phosphate promoted NADPH-dependent activity by stabilising the unstable tetrameric form produced from the hexadecamer by the thiol.These phenomena are discussed in relation to a general mechanism for the in vivo promotion of NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase activity.     

Improved production of 2-keto-3-deoxy-d-glycero-galactononulosonic acid (KDN) using FastPrep-CLEAs

FastPrep cross-linked enzyme aggregates of N-acetylneuraminate aldolase from Staphylococcus carnosus (ScNAL-FpCLEAs) were prepared in order to improve the synthesis of 2-keto-3-deoxy-d-glycero-galactononulosonic acid (KDN), an important building block for therapeutic glycolipids and a possible marker for human prostate cancer. ScNAL-FpCLEAs showed improved thermostability compared with the free enzyme, doubling its half-life at 60 °C. When the effect of substrate ratio (pyruvate:d-mannose) and temperature on the yield of KDN was studied at its optimum pH (pH 7.0), 90% conversion in only 8 h was reached in the presence of 0.6 M d-mannose and 1.2 M pyruvate at 37 °C. This is the highest conversion described to date for enzymatic KDN synthesis. In addition, ScNAL-FpCLEAs exhibited enhanced catalytic activity and stability and could be recycled 10 times with no loss of activity. These results suggest the biotechnological potential of using FastPrepCLEAs to obtain valuable biocatalysts.