Adaptive evolution of the Streptococcus pyogenes regulatory aldolase LacD.1.

In the human-pathogenic bacterium Streptococcus pyogenes, the tagatose bisphosphate aldolase LacD.1 likely originated through a gene duplication event and was adapted to a role as a metabolic sensor for regulation of virulence gene transcription. Although LacD.1 retains enzymatic activity, its ancestral metabolic function resides in the LacD.2 aldolase, which is required for the catabolism of galactose. In this study, we compared these paralogous proteins to identify characteristics correlated with divergence and novel function. Surprisingly, despite the fact that these proteins have identical active sites and 82% similarity in amino acid sequence, LacD.1 was less efficient at cleaving both fructose and tagatose bisphosphates. Analysis of kinetic properties revealed that LacD.1's adaptation was associated with a decrease in k(cat) and an increase in K(m). Construction and analysis of enzyme chimeras indicated that non-active-site residues previously associated with the variable activities of human aldolase isoenzymes modulated LacD.1's affinity for substrate. Mutant LacD.1 proteins engineered to have LacD.2-like levels of enzymatic efficiency lost the ability to function as regulators, suggesting that an alteration in efficiency was required for adaptation. In competition under growth conditions that mimic a deep-tissue environment, LacD.1 conferred a significant gain in fitness that was associated with its regulatory activity. Taken together, these data suggest that LacD.1's adaptation represents a form of neofunctionalization in which duplication facilitated the gain of regulatory function important for growth in tissue and pathogenesis.

Insights from crystal structures into the opposite effects on RNA affinity caused by the S- and R-6'-methyl backbone modifications of 3'-fluoro hexitol nucleic acid.

Locked nucleic acid (LNA) analogues with 2',4'-bridged sugars show promise in antisense applications. S-5'-Me-LNA has high RNA affinity, and modified oligonucleotides show weakened immune stimulation in vivo. Conversely, an R-5'-methyl group dramatically lowers RNA affinity. To test the effects of S- and R-6'-methyl groups on 3'-fluoro hexitol nucleic acid (FHNA) stability, we synthesized S- and R-6'-Me-FHNA thymidine and incorporated them into oligo-2'-deoxynucleotides. As with LNA, S-6'-Me is stabilizing whereas R-6'-Me is destabilizing. Crystal structures of 6'-Me-FHNA-modified DNAs explain the divergent consequences for stability and suggest convergent origins of these effects by S- and R-6'-Me (FHNA) [-5'-Me (LNA and RNA)] substituents.

Structural insights into the substrate binding and stereoselectivity of giardia fructose-1,6-bisphosphate aldolase.

Giardia lamblia fructose-1,6-bisphosphate aldolase (FBPA) is a member of the class II zinc-dependent aldolase family that catalyzes the cleavage of d-fructose 1,6-bisphosphate (FBP) into dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (G3P). In addition to the active site zinc, the catalytic apparatus of FBPA employs an aspartic acid, Asp83 in the G. lamblia enzyme, which when replaced with an alanine residue renders the enzyme inactive. A comparison of the crystal structures of D83A FBPA in complex with FBP and of wild-type FBPA in the unbound state revealed a substrate-induced conformational transition of loops in the vicinity of the active site and a shift in the location of Zn(2+). When FBP binds, the Zn(2+) shifts up to 4.6 A toward the catalytic Asp83, which brings the metal within coordination distance of the Asp83 carboxylate group. In addition, the structure of wild-type FBPA was determined in complex with the competitive inhibitor d-tagatose 1,6-bisphosphate (TBP), a FBP stereoisomer. In this structure, the zinc binds in a site close to that previously seen in the structure of FBPA in complex with phosphoglycolohydroxamate, an analogue of the postulated DHAP ene-diolate intermediate. Together, the ensemble of structures suggests that the zinc mobility is necessary to orient the Asp83 side chain and to polarize the substrate for proton transfer from the FBP C(4) hydroxyl group to the Asp83 carboxyl group. In the absence of FBP, the alternative zinc position is too remote for coordinating the Asp83. We propose a modification of the catalytic mechanism that incorporates the novel features observed in the FBPA-FBP structure. The mechanism invokes coordination and coplanarity of the Zn(2+) with the FBP's O-C(3)-C(4)-O group concomitant with coordination of the Asp83 carboxylic group. Catalysis is accompanied by movement of Zn(2+) to a site coplanar with the O-C(2)-C(3)-O group of the DHAP. glFBPA exhibits strict substrate specificity toward FBP and does not cleave TBP. The active sites of FBPAs contain an aspartate residue equivalent to Asp255 of glFBPA, whereas tagatose-1,6-bisphosphate aldolase contains an alanine in this position. We and others hypothesized that this aspartic acid is a likely determinant of FBP versus TBP specificity. Replacement of Asp255 with an alanine resulted in an enzyme that possesses double specificity, now cleaving TBP (albeit with low efficacy; k(cat)/K(m) = 80 M(-1) s(-1)) while maintaining activity toward FBP at a 50-fold lower catalytic efficacy compared with that of wild-type FBPA. The collection of structures and sequence analyses highlighted additional residues that may be involved in substrate discrimination.

Stereoselectivity of fructose-1,6-bisphosphate aldolase in Thermus caldophilus.

It was recently established that fructose-1,6-bisphosphate (FBP) aldolase (FBA) and tagatose-1,6-bisphosphate (TBP) aldolase (TBA), two class II aldolases, are highly specific for the diastereoselective synthesis of FBP and TBP from glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), respectively. In this paper, we report on a FBA from the thermophile Thermus caldophilus GK24 (Tca) that produces both FBP and TBP from C(3) substrates. Moreover, the FBP:TBP ratio could be adjusted by manipulating the concentrations of G3P and DHAP. This is the first native FBA known to show dual diastereoselectivity among the FBAs and TBAs characterized thus far. To explain the behavior of this enzyme, the X-ray crystal structure of the Tca FBA in complex with DHAP was determined at 2.2A resolution. It appears that as a result of alteration of five G3P binding residues, the substrate binding cavity of Tca FBA has a greater volume than those in the Escherichia coli FBA-phosphoglycolohydroxamate (PGH) and TBA-PGH complexes. We suggest that this steric difference underlies the difference in the diastereoselectivities of these class II aldolases.

Hexitol nucleic acid-containing aptamers are efficient ligands of HIV-1 TAR RNA.

The transactivation responsive element (TAR) plays a crucial role in the transcription of the HIV-1 genome upon specific binding of the viral protein Tat and cellular proteins. We have previously identified a RNA hairpin aptamer forming a stable and specific kissing complex with TAR RNA (Ducongé, F., and Toulmé, J. J. (1999) RNA 5, 1605-1614). We chemically modified this aptamer with hexitol nucleic acid (HNA) residues. We demonstrate that a fully HNA-modified aptamer is a poor ligand but, in contrast, mixmers containing both HNA and unmodified RNA nucleotides display interesting properties. Two HNA-RNA mixmers bind to TAR with an equilibrium dissociation constant in the low-nanomolar range and show a reduced nuclease sensitivity. In addition, they show a moderate dependence on magnesium ions for binding to TAR. These HNA-RNA mixmers are able to inhibit transactivation of transcription in an in vitro assay.

Modifying the stereochemistry of an enzyme-catalyzed reaction by directed evolution.

Aldolases have potential as tools for the synthesis of stereochemically complex carbohydrates. Here, we show that directed evolution can be used to alter the stereochemical course of the reaction catalyzed by tagatose-1,6-bisphosphate aldolase. After three rounds of DNA shuffling and screening, the evolved aldolase showed an 80-fold improvement in k(cat)/K(m) toward the non-natural substrate fructose 1,6-bisphosphate, resulting in a 100-fold change in stereospecificity. (31)P NMR spectroscopy was used to show that, in the synthetic direction, the evolved aldolase catalyzes the formation of carbon-carbon bonds with unnatural diastereoselectivity, where the >99:<1 preference for the formation of tagatose 1,6-bisphosphate was switched to a 4:1 preference for the diastereoisomer, fructose 1,6-bisphosphate. This demonstration is of considerable significance to synthetic chemists requiring efficient syntheses of complex stereoisomeric products, such as carbohydrate mimetics.

Two class II D-tagatose-bisphosphate aldolases from enteric bacteria.

Escherichia coli, Salmonella enterica, Klebsiella pneumoniaeand Klebsiella oxytocawere found to contain two D-tagatose 1,6-bisphosphate (TagBP)-specific aldolases involved in catabolism of galactitol (genes gatY gatZ) and of N-acetyl-galactosamine and D-galactosamine (genes kbaY kbaZ,also called agaY agaZ). The two aldolases were closely related (> or = 53.8% identical amino acids) and could substitute for each other in vivo. The catalytic subunits GatY or KbaY alone were sufficient to show aldolase activity. Although substantially shorter than other aldolases (285 amino acids, instead of 358 and 349 amino acids), these subunits contained most or all of the residues that have been identified as essential in substrate/product recognition and catalysis for class II aldolases. In contrast to these, both aldolases required subunits GatZ or KbaZ (420 amino acids) for full activity and for good in vivo and in vitro stability. The Z subunits alone did not show any aldolase activity. Close relatives of these new TagBP aldolases were found in several gram-negative and gram-positive bacteria, e.g., Streptomyces coelicolor.

A comparative study on diurnal changes in metabolite levels in the leaves of three crassulacean acid metabolism (CAM) species, Ananas comosus, Kalanchoë daigremontiana and K. pinnata.

A comparative study on diurnal changes in metabolite levels associated with crassulacean acid metabolism (CAM) in the leaves of three CAM species, Ananas comosus (pineapple), a hexose-utilizing species, and Kalanchoë daigremontiana and K. pinnata, two starch-utilizing species, were made. All three CAM species showed a typical feature of CAM with nocturnal malate increase. In the two Kalanchoë species, isocitrate levels were higher than citrate levels; the reverse was the case in pineapple. In the two Kalanchoë species, a small nocturnal citrate increase was found and K. daigremontiana showed a small nocturnal isocitrate increase. Glucose 6-phosphate (G-6-P), fructose 6-phosphate (F-6-P) and glucose 1-phosphate (G-1-P) levels in the three CAM species rose rapidly during the first part of the dark period and decreased during the latter part of the dark period. The levels of the metabolites also decreased during the first 3 h of the light period, then, remained little changed through the rest of the light period. Absolute levels of G-6-P, F-6-P and G-1-P were higher in pineapple than in the two Kalanchoë species. Fructose 1,6-bisphosphate (F-1,6-P(2)) levels in the three CAM species increased during the dark period, then dramatically decreased during the first 3 h of the light period and remained unchanged through the rest of the light period. The extent of nocturnal F-1,6-P(2) increase was far greater in the two Kalanchoë species than in pineapple. Absolute levels of F-1,6-P(2) were higher in the two Kalanchoë species than in pineapple, especially during dark period. Diurnal changes in oxaloacetate (OAA), pyruvate (Pyr) and phosphoenolpyruvate (PEP) levels in the three CAM species were similar.

Exploring substrate binding and discrimination in fructose1, 6-bisphosphate and tagatose 1,6-bisphosphate aldolases.

Fructose 1,6-bisphosphate aldolase catalyses the reversible condensation of glycerone-P and glyceraldehyde 3-phosphate into fructose 1,6-bisphosphate. A recent structure of the Escherichia coli Class II fructose 1,6-bisphosphate aldolase [Hall, D.R., Leonard, G.A., Reed, C.D., Watt, C.I., Berry, A. & Hunter, W.N. (1999) J. Mol. Biol. 287, 383-394] in the presence of the transition state analogue phosphoglycolohydroxamate delineated the roles of individual amino acids in binding glycerone-P and in the initial proton abstraction steps of the mechanism. The X-ray structure has now been used, together with sequence alignments, site-directed mutagenesis and steady-state enzyme kinetics to extend these studies to map important residues in the binding of glyceraldehyde 3-phosphate. From these studies three residues (Asn35, Ser61 and Lys325) have been identified as important in catalysis. We show that mutation of Ser61 to alanine increases the Km value for fructose 1, 6-bisphosphate 16-fold and product inhibition studies indicate that this effect is manifested most strongly in the glyceraldehyde 3-phosphate binding pocket of the active site, demonstrating that Ser61 is involved in binding glyceraldehyde 3-phosphate. In contrast a S61T mutant had no effect on catalysis emphasizing the importance of an hydroxyl group for this role. Mutation of Asn35 (N35A) resulted in an enzyme with only 1.5% of the activity of the wild-type enzyme and different partial reactions indicate that this residue effects the binding of both triose substrates. Finally, mutation of Lys325 has a greater effect on catalysis than on binding, however, given the magnitude of the effects it is likely that it plays an indirect role in maintaining other critical residues in a catalytically competent conformation. Interestingly, despite its proximity to the active site and high sequence conservation, replacement of a fourth residue, Gln59 (Q59A) had no significant effect on the function of the enzyme. In a separate study to characterize the molecular basis of aldolase specificity, the agaY-encoded tagatose 1,6-bisphosphate aldolase of E. coli was cloned, expressed and kinetically characterized. Our studies showed that the two aldolases are highly discriminating between the diastereoisomers fructose bisphosphate and tagatose bisphosphate, each enzyme preferring its cognate substrate by a factor of 300-1500-fold. This produces an overall discrimination factor of almost 5 x 105 between the two enzymes. Using the X-ray structure of the fructose 1,6-bisphosphate aldolase and multiple sequence alignments, several residues were identified, which are highly conserved and are in the vicinity of the active site. These residues might potentially be important in substrate recognition. As a consequence, nine mutations were made in attempts to switch the specificity of the fructose 1,6-bisphosphate aldolase to that of the tagatose 1,6-bisphosphate aldolase and the effect on substrate discrimination was evaluated. Surprisingly, despite making multiple changes in the active site, many of which abolished fructose 1, 6-bisphosphate aldolase activity, no switch in specificity was observed. This highlights the complexity of enzyme catalysis in this family of enzymes, and points to the need for further structural studies before we fully understand the subtleties of the shaping of the active site for complementarity to the cognate substrate.

Antimalarial antisense activity of hexitol nucleic acids.

Antisense oligonucleotides and ribozymes have shown promise both as antimalarial agents and as tools for identifying genes vital for parasite survival. This task is urgent due to the ineffectiveness of current drug regimes on the most virulent human malarial parasite, Plasmodium falciparum. The development of new ways to modify and/or protect conventional phosphodiester oligonucleotides to improve nuclease resistance is also important. We assessed the effect of antisense oligonucleotides containing phosphorylated anhydrohexitols in suppressing the growth of P. falciparum in culture. The modified oligonucleotides were able to inhibit parasite growth in a sequence-specific manner, but not as well as the phosphorothioated antisense oligonucleotides, which are effective antimalarials at submicromolar concentrations. Two reasons are suggested: the absence of RNase H activation and differences in membrane transport.

Contrasting effects of the protein kinase C inhibitor, staurosporine, on cytokine and phorbol ester stimulation of fructose 2,6-bisphosphate and prostaglandin E production by fibroblasts in vitro. Comparative studies using interleukin-1 alpha, tumour necrosis factor alpha, transforming growth factor beta, interferon-gamma and 12-O-tetradecanoylphorbol 13-acetate.

It is known that both interleukin-1 alpha (IL-1 alpha) and 12-O-tetradecanoylphorbol 13-acetate (TPA) promote increases in intracellular levels of the glycolytic regulatory metabolite fructose 2,6-bisphosphate [Fru(2,6)P2] and in the production of prostaglandin E (PGE) by subcultured rheumatoid synovial cells (RSC) and human dermal fibroblasts in vitro. We report here that the protein kinase C inhibitor staurosporine enhanced the IL-1 alpha-induced increase in [Fru(2,6)P2] and PGE production by RSC, whereas in similar concentrations (3-30 nM) this inhibitor decreased the TPA-induced stimulation of these parameters. Staurosporine produced a similar enhancement of the response to IL-1 alpha by normal human dermal fibroblasts. The increased PGE production provoked by tumour necrosis factor alpha (TNF alpha) in RSC was also augmented by staurosporine, but, in contrast, the increases in cellular [Fru(2,6)P2] induced by transforming growth factor beta (TGF beta) and interferon-gamma (IFN-gamma) were diminished. Thus the protein kinase C inhibitor staurosporine discriminates not only between the effects produced by IL-1 alpha and TPA, but also between those of IL-1 alpha and two other cytokines (but not between IL-1 alpha and TNF alpha). These findings suggest that IL-1 alpha and probably TNF alpha act via an intracellular mechanism different from that mediating the action of TPA, TGF-beta and IFN-gamma, and provide evidence that staurosporine is capable of amplifying the IL-1 signal.

Effect of streptozotocin diabetes on the glycolytic flux and on fructose 2,6-bisphosphate levels in isolated rat enterocytes.

In epithelial cells isolated from rat small intestine and incubated in the presence of 1 mM glucose, streptozotocin-induced diabetes reduced, by 46 and 29%, respectively, the rates of both glucose utilization and L-lactate formation. These effects were accompanied by a significant decrease of enterocyte fructose 2,6-bisphosphate concentration (about 50%) and of the glycolytic flux through the reaction catalyzed by 6-phosphofructo 1-kinase. The diminution of enterocyte fructose 2,6-bisphosphate levels caused by diabetes occurred in spite of an increase of hexose 6-phosphate concentration, and was associated with a reduction in the amount of active form of 6-phosphofructo 2-kinase; total activity of this enzyme was not significantly modified. Diabetes also caused an acceleration in the rate of 3-O-methyl-D-(14C) glucose uptake and increased hexokinase activity in enterocytes. Lactate dehydrogenase, pyruvate kinase and 6-phosphofructo 1-kinase activities were not found to be significantly different in epithelial cells isolated from control or diabetic animals. Our results indicate that a reduction of the glycolytic flux in enterocytes could collaborate to increase intestinal glucose absorption in the diabetic state.

Fructose-1,6-bisphosphate-activated pyruvate kinase from Escherichia coli. Nature of bonds involved in the allosteric mechanism.

The allosteric properties of the fructose-1,6-bis-phosphate-activated pyruvate kinase from Escherichia coli were examined in the presence of a number of fructose bisphosphate analogues, as well as of increased ionic strength (NaCl) and of the hydrogen-bond-breaking agent, formamide. Fructose 2,6-bisphosphate, ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate gave allosteric activation (additive to that of fructose 1,6-bisphosphate). Formamide always decreased Vmax, but left unchanged the Km for phosphoenolpyruvate, while it decreased the concentration of fructose bisphosphate required to give half-maximal activity (K0.5). NaCl increased the K0.5 for both phosphoenolpyruvate and fructose bisphosphate, leaving Vmax unchanged. These results are consistent with ionic binding of fructose bisphosphate through phosphates and with a critical role of hydrogen bonds in stabilizing both the inactive and the active enzyme conformers.

The effects of various anesthetics on tissue levels of fructose-2,6-bisphosphate in rats.

We report that the short-term use of various anesthetic agents prior to decapitation causes alteration of the levels of fructose-2,6-bisphosphate in kidney, brain, heart, muscle, and liver. These data indicate that even light anesthesia can not be used when levels of this metabolite are to be determined. Also, it appears that the use of any of these anesthetics can profoundly alter glucose utilization in many tissues.

Fructose 2,6-bisphosphate and glycolytic flux in skeletal muscle of swimming frog.

Glycolytic flux in skeletal muscle is controlled by 6-phosphofructokinase but how this is achieved is controversial. Brief exercise (swimming) in frogs caused a dramatic increase in the phosphofructokinase activator, fructose 2,6-bisphosphate, in working muscle. The kinetics of phosphofructokinase suggest that in resting muscle, the enzyme is inhibited by ATP plus citrate and that the increase in fructose 2,6-bisphosphate is part of the mechanism to activate phosphofructokinase when exercise begins. When exercise was sustained, fructose 2,6-bisphosphate in muscle was decreased as was the rate of lactate accumulation. Glycolytic flux and the content of fructose 2,6-bisphosphate appear to be closely correlated in working frog muscle in vivo.

2',5' A synthetase: allosteric activation by fructose 1,6-bisphosphate.

Fructose 1,6-bisphosphate (fru-1,6-P2), but not other glycolytic intermediates, activates highly purified 2',5' A synthetases from rabbit reticulocyte lysates and from 2',5'-ADP-agarose purified extracts of interferon-treated HeLa cells without the addition of dsRNA. The 2',5' A was structurally and biologically identical to authentic 2',5' A. Micrococcal nuclease inhibited the activation of 2',5' A synthetase by poly(I)-poly(C), but did not affect activation by fru-1,6-P2. Addition of fru-1,6-P2 aldolase prevented the activation of 2',5' A synthetase by fru-1,6-P2.

Fructose-1,6-diphosphate counteracts ethanol-stimulated calcium uptake in isolated BHK cells.

Ethanol increases the uptake of 45Ca by isolated baby hamster kidney (BHK) cells in vitro. The effect is dependent on ethanol and 45Ca++ concentration and on the incubation time. Fructose-1,6-diphosphate (FDP) added at different concentration during the pre-incubation exerts a protective effect through a membrane-stabilizing action which is consistent with its in vivo anti-alcohol activity documented in previous studies.

Fructose-2,6-bisphosphate in control of hepatic gluconeogenesis. From metabolites to molecular genetics.

Hormonal regulation of hepatic gluconeogenic pathway flux is brought about by phosphorylation/dephosphorylation and control of gene expression of several key regulatory enzymes. Regulation by cAMP-dependent phosphorylation occurs at the level of pyruvate kinase and 6-phosphofructo-2-kinase (6PF-1-K)/fructose-2,6-bisphosphatase (Fru-2,6-P2ase). The latter is a unique bifunctional enzyme that catalyzes both the synthesis and degradation of fructose-2,6-bisphosphate (Fru-2,6-P2), which is an activator of 6PF-1-K and an inhibitor of Fru-1,6-P2ase. The bifunctional enzyme is a homodimer whose activities are regulated by cAMP-dependent protein kinase-catalyzed phosphorylation at a single NH2-terminal seryl residue/subunit, which results in activation of the Fru-2,6-P2ase and inhibition of the PF-1-K reactions. Hormone-mediated changes in the phosphorylation state of the bifunctional enzyme are responsible for acute regulation of Fru-2,6-P2 levels. 6PF-2-K/Fru-2,6-P2ase thus provides a switching mechanism between glycolysis and gluconeogenesis in mammalian liver. Pyruvate kinase is regulated by both phosphorylation and allosteric effectors. Fru-1,6-P2, an allosteric activator, also inhibits cAMP-dependent enzyme phosphorylation, and its steady-state concentration is indirectly determined by the level of Fru-2,6-P2. Therefore, acute regulation of both pyruvate kinase and the bifunctional enzyme provide coordinated control at both the pyruvate/phosphoenolpyruvate and Fru-6-P/Fru-1,6-P2 substrate cycles. The Fru-2,6-P2 system is also subject to complex multihormonal long-term control through regulation of 6 PF-2-K/Fru-2,6-P2ase gene expression. Glucocorticoids are the major factor in turning on this gene in liver, but insulin is also a positive effector. cAMP prevents the effects of glucocorticoids and insulin. Although Fru-2,6-P2 plays a key role in the regulation of carbon flux in the gluconeogenic pathway, the regulation of this flux depends on several factors and regulation of other key enzymes whose importance varies depending on the dietary and hormonal status of the animal. Molecular cloning of the cDNA encoding PF-2-K/Fru-2,6-P2ase has elucidated its structure and permitted analysis of its evolutionary origin as well as its tissue distribution and control of its gene expression. The rat liver and skeletal muscle isoforms arose by alternative splicing of a single gene. The muscle form differs from the liver form only at the NH2-terminal and does not have a cAMP-dependent protein kinase phosphorylation site. The hepatic enzyme subunit consists of 470 amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)

Fructose-1,6-bisphosphate reduces ATP loss from hypoxic astrocytes.

Hypoxia caused injury and metabolic dysfunction of astrocytes, as indicated by a time-dependent loss of lactate dehydrogenase (LDH) activity and ATP content. The combination of 3.5 mM fructose-1,6-bisphosphate (FBP) and 7.5 mM glucose (GLC) reduced the decrease of ATP and prevented the loss of LDH. These data indicate that the combination of GLC + FBP protects astrocytes from hypoxia. The results also suggest that the maintainance of ATP concentration is the mechanism by which FBP prevents hypoxic injury.