The phosphate moiety of phosphoenolpyruvate does NOT contribute to allosteric regulation of liver pyruvate kinase by fructose-1,6-bisphosphate✝.

A linked-function theory for allostery allows for a differentiation between those protein-ligand interactions that contribute the most to ligand binding and those protein-ligand interactions that contribute to the allosteric mechanism. This potential distinction is the basis for analogue studies used to determine which chemical moieties on the allosteric effector contribute to allostery. Although less recognized, the same separation of functions is possible for substrate-enzyme interactions. When evaluating allosteric regulation in human liver pyruvate kinase, the use of a range of monovalent cations (K+, NH4+, Rb+, Cs+, cyclohexylammonium+ and Tris+) altered substrate (phosphoenolpyruvate; PEP) affinity, but maintained similar allosteric responses to the allosteric activator, fructose-1,6-bisphosphate (Fru-1,6-BP). Because crystal structures indicate that the active site monovalent cation interacts directly with the phosphate moiety of the bound PEP substrate, we questioned if the phosphate moiety might contribute to substrate binding, but not to the allosteric mechanism. Here, we demonstrate that the binding of oxalate, a non-phosphorylated substrate/product analogue, is allosterically enhanced by Fru-1,6-BP. That observation is consistent with the concept that the phosphate moiety of PEP is not required for the allosteric function, even though that moiety likely contributes to determining substrate affinity.

Mechanistic insights into the allosteric regulation of bacterial ADP-glucose pyrophosphorylases.

ADP-glucose pyrophosphorylase (AGPase) controls bacterial glycogen and plant starch biosynthetic pathways, the most common carbon storage polysaccharides in nature. AGPase activity is allosterically regulated by a series of metabolites in the energetic flux within the cell. Very recently, we reported the first crystal structures of the paradigmatic AGPase from Escherichia coli (EcAGPase) in complex with its preferred physiological negative and positive allosteric regulators, adenosine 5'-monophosphate (AMP) and fructose 1,6-bisphosphate (FBP), respectively. However, understanding the molecular mechanism by which AMP and FBP allosterically modulates EcAGPase enzymatic activity still remains enigmatic. Here we found that single point mutations of key residues in the AMP-binding site decrease its inhibitory effect but also clearly abolish the overall AMP-mediated stabilization effect in wild-type EcAGPase. Single point mutations of key residues for FBP binding did not revert the AMP-mediated stabilization. Strikingly, an EcAGPase-R130A mutant displayed a dramatic increase in activity when compared with wild-type EcAGPase, and this increase correlated with a significant increment of glycogen content in vivo The crystal structure of EcAGPase-R130A revealed unprecedented conformational changes in structural elements involved in the allosteric signal transmission. Altogether, we propose a model in which the positive and negative energy reporters regulate AGPase catalytic activity via intra- and interprotomer cross-talk, with a "sensory motif" and two loops, RL1 and RL2, flanking the ATP-binding site playing a significant role. The information reported herein provides exciting possibilities for industrial/biotechnological applications.

The Simple and Unique Allosteric Machinery of Thermus caldophilus Lactate Dehydrogenase : Structure-Function Relationship in Bacterial Allosteric LDHs.

Many bacterial L-lactate dehydrogenases (LDH) are allosteric enzymes, and usually activated by fructose 1,6-bisphosphate (FBP) and often also by substrate pyruvate. The active and inactive state structures demonstrate that Thermus caldophilus, Lactobacillus casei, and Bifidobacterium longum LDHs consistently undergo allosteric transition according to Monod-Wyman-Changeux model, where the active (R) and inactive (T) states of the enzymes coexist in an allosteric equilibrium (pre-existing equilibrium) independently of allosteric effectors. The three enzymes consistently take on open and closed conformations of the homotetramers for the T and R states, coupling the quaternary structural changes with the structural changes in binding sites for substrate and FBP though tertiary structural changes. Nevertheless, the three enzymes undergo markedly different structural changes from one another, indicating that there is a high variety in the allosteric machineries of bacterial LDHs. L. casei LDH undergoes the largest quaternary structural change in the three enzymes, and regulates its catalytic activity though a large linkage frame for allosteric motion. In contrast, T. caldophilus LDH exhibits the simplest allosteric motion in the three enzymes, involving a simple mobile structural core for the allosteric motion. TcLDH likely mediates its allosteric equilibrium mostly through electrostatic repulsion within the protein molecule, providing an insight for regulation machineries in bacterial allosteric LDHs.

Fructose 1,6-bisphosphate aldolase/phosphatase may be an ancestral gluconeogenic enzyme.

Most archaeal groups and deeply branching bacterial lineages harbour thermophilic organisms with a chemolithoautotrophic metabolism. They live at high temperatures in volcanic habitats at the expense of inorganic substances, often under anoxic conditions. These autotrophic organisms use diverse carbon dioxide fixation mechanisms generating acetyl-coenzyme A, from which gluconeogenesis must start. Here we show that virtually all archaeal groups as well as the deeply branching bacterial lineages contain a bifunctional fructose 1,6-bisphosphate (FBP) aldolase/phosphatase with both FBP aldolase and FBP phosphatase activity. This enzyme is missing in most other Bacteria and in Eukaryota, and is heat-stabile even in mesophilic marine Crenarchaeota. Its bifunctionality ensures that heat-labile triosephosphates are quickly removed and trapped in stabile fructose 6-phosphate, rendering gluconeogenesis unidirectional. We propose that this highly conserved, heat-stabile and bifunctional FBP aldolase/phosphatase represents the pace-making ancestral gluconeogenic enzyme, and that in evolution gluconeogenesis preceded glycolysis.

Distinguishing the interactions in the fructose 1,6-bisphosphate binding site of human liver pyruvate kinase that contribute to allostery.

In the study of allosteric proteins, understanding which effector-protein interactions contribute to allosteric activation is important both for designing allosteric drugs and for understanding allosteric mechanisms. The antihyperglycemic target, human liver pyruvate kinase (hL-PYK), binds its allosteric activator, fructose 1,6-bisphosphate (Fru-1,6-BP), such that the 1'-phosphate interacts with side chains of Arg501 and Trp494 and the 6'-phosphate interacts with Thr444, Thr446, Ser449 (i.e., the 444-449 loop), and Ser531. Additionally, backbone atoms from the 527-533 loop interact with a sugar ring hydroxyl and the two effector phosphate moieties. An effector analogue series indicates that only one phosphate on the sugar is required for activation. However, singly phosphorylated sugars, including Fru-1-P and Fru-6-P, bind with a Kix in the range of 0.07-1 mM. The second phosphate of Fru-1,6-BP causes tight effector binding, because this native effector has a Kix of 0.061 µM. Glucose 1,6-bisphosphate and ribulose 1,5-bisphosphate bind in the 0.07-1 mM range. The contrast with a higher Fru-1,6-BP binding indicates specificity for the fructose sugar conformation. Site-directed random mutagenesis at each residue that contacts bound Fru-1,6-BP showed that a negative charge introduced at position 531 mimics allosteric activation, even in the absence of Fru-1,6-BP. Collectively, analogue and mutagenesis studies are consistent with the 527-533 loop playing a key role in allosteric function. Deletion mutations that shortened the 527-533 loop were expected to prevent formation of hydrogen bonds between backbone atoms on the loop and Fru-1,6-BP. Indeed, Fru-1,6-BP did not activate these loop-shortened mutant proteins. Previous structural comparisons of M1-PYK and M2-PYK indicate that the 527-533 loop makes interactions across a subunit interface when an activator is not present. Mutating the hL-PYK subunit interface interactions among Trp527, Arg528, and Asp499 mimics allosteric activation. Considered with published structures, these results are consistent with (1) the two phosphates of Fru-1,6-BP docking to Arg501/Trp494 and the 444-449 loop, respectively, and (2) the formation of hydrogen bonds among Fru-1,6-BP and backbone atoms of the 527-533 loop pulling this loop away from the subunit interface, which results in breaking of the Trp527-Arg528-Asp499 interactions to elicit an allosteric response.

The core of allosteric motion in Thermus caldophilus L-lactate dehydrogenase.

For Thermus caldophilus L-lactate dehydrogenase (TcLDH), fructose 1,6-bisphosphate (FBP) reduced the pyruvate S(0.5) value 10(3)-fold and increased the V(max) value 4-fold at 30 °C and pH 7.0, indicating that TcLDH has a much more T state-sided allosteric equilibrium than Thermus thermophilus L-lactate dehydrogenase, which has only two amino acid replacements, A154G and H179Y. The inactive (T) and active (R) state structures of TcLDH were determined at 1.8 and 2.0 Å resolution, respectively. The structures indicated that two mobile regions, MR1 (positions 172-185) and MR2 (positions 211-221), form a compact core for allosteric motion, and His(179) of MR1 forms constitutive hydrogen bonds with MR2. The Q4(R) mutation, which comprises the L67E, H68D, E178K, and A235R replacements, increased V(max) 4-fold but reduced pyruvate S(0.5) only 5-fold in the reaction without FBP. In contrast, the P2 mutation, comprising the R173Q and R216L replacements, did not markedly increase V(max), but 10(2)-reduced pyruvate S(0.5), and additively increased the FBP-independent activity of the Q4(R) enzyme. The two types of mutation consistently increased the thermal stability of the enzyme. The MR1-MR2 area is a positively charged cluster, and its center approaches another positively charged cluster (N domain cluster) across the Q-axis subunit interface by 5 Å, when the enzyme undergoes the T to R transition. Structural and kinetic analyses thus revealed the simple and unique allosteric machinery of TcLDH, where the MR1-MR2 area pivotally moves during the allosteric motion and mediates the allosteric equilibrium through electrostatic repulsion within the protein molecule.

Subunit interactions in pig-kidney fructose-1,6-bisphosphatase: binding of substrate induces a second class of site with lowered affinity and catalytic activity.

BACKGROUND: Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, Fru-2,6-P2 and by high concentrations of its substrate Fru-1,6-P2. The mechanism that produces substrate inhibition continues to be obscure. METHODS: Four types of experiments were used to shed light on this: (1) kinetic measurements over a very wide range of substrate concentrations, subjected to detailed statistical analysis; (2) fluorescence studies of mutants in which phenylalanine residues were replaced by tryptophan; (3) effect of Fru-2,6-P2 and Fru-1,6-P2 on the exchange of subunits between wild-type and Glu-tagged oligomers; and (4) kinetic studies of hybrid forms of the enzyme containing subunits mutated at the active site residue tyrosine-244. RESULTS: The kinetic experiments with the wild-type enzyme indicate that the binding of Fru-1,6-P2 induces the appearance of catalytic sites with lower affinity for substrate and lower catalytic activity. Binding of substrate to the high-affinity sites, but not to the low-affinity sites, enhances the fluorescence emission of the Phe219Trp mutant; the inhibitor, Fru-2,6-P2, competes with the substrate for the high-affinity sites. Binding of substrate to the low-affinity sites acts as a "stapler" that prevents dissociation of the tetramer and hence exchange of subunits, and results in substrate inhibition. CONCLUSIONS: Binding of the first substrate molecule, in one dimer of the enzyme, produces a conformational change at the other dimer, reducing the substrate affinity and catalytic activity of its subunits. GENERAL SIGNIFICANCE: Mimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia.

Enzyme Substrate Specificity Conferred by Distinct Conformational Pathways.

Substrate recognition is one of the hallmarks of enzyme catalysis. Enzyme conformational changes have been linked to selectivity between substrates with little direct evidence. Aldolase, a glycolytic enzyme, must distinguish between two physiologically important substrates, fructose 1-phosphate and fructose 1,6-bisphosphate, and provides an excellent model system for the study of this question. Previous work has shown that isozyme specific residues (ISRs) distant from the active site are responsible for kinetic distinction between these substrates. Notably, most of the ISRs reside in a cluster of five surface α-helices, and the carboxyl-terminal region (CTR), and cooperative interactions among these helices have been demonstrated. To test the hypothesis that conformational changes are at the root of these changes, single surface-cysteine variants were created with the cysteine located on helices of the cluster and CTR. This allowed for site-specific labeling with an environmentally sensitive fluorophore, and subsequent monitoring of conformational changes by fluorescence emission spectrophotometry. These labeled variants revealed different spectra in the presence of saturating amounts of each substrate, which suggested the occurrence of different conformations. Emission spectra collected at various substrate concentrations showed a concentration dependence of the fluorescence spectra, consistent with binding events. Lastly, stopped-flow fluorescence spectrophotometry showed that the rate of these fluorescence changes was on the same time-scale as catalysis, thus suggesting a link between the different fluorescence changes and events during catalysis. On the basis of these results, we propose that different conformational changes may be a common mechanism for dictating substrate specificity in other enzymes with multiple substrates.

Effect of fructose 1,6-bisphosphate on the iron redox state relating to the generation of reactive oxygen species.

Role of fructose 1,6-bisphosphate-mediated iron oxidation in the generation of reactive oxygen species was analyzed. Aconitase the most sensitive enzyme to oxidative stress was inactivated potently by fructose 1,6-bisphosphate in the presence of ferrous ion, and further by ADP and PEP to a lesser extent. The inactivation requires cyanide, suggesting that the superoxide radical is responsible for the inactivation. Addition of ascorbic acid and dithiothreitol prevented aconitase from the inactivation. Fructose 1,6-bisphosphate, ADP and PEP stimulated the oxidation of ferrous ion causing one-electron reduction of oxygen molecule. Superoxide radical formed with iron oxidation participates in the oxidative inactivation of aconitase and the citric acid cycle, resulting in the induction of the Crabtree effect, that is, high glucose-mediated inhibition of oxidative metabolism in mitochondria.

Phosphatase-mimicking Zr@PDA nanozyme with excellent dispersion stability for the detection of fructose 1,6-diphosphate.

Zr4+-doped polydopamine (Zr@PDA) nanozyme with phosphatase-like activity was synthesized by a one-pot hydrothermal method for the first time. Compared with previous representative phosphatase-mimicking nanozymes (i.e., CeO2 NPs, ZrO2 NPs and UiO-66), Zr@PDA not only exhibited higher dispersion stability in water, but also higher catalytical efficiency. Kcat/Km of Zr@PDA is 35 and 12 times that of UiO-66 and ZrO2 NPs, respectively, which would endow the Zr@PDA-based analytical methods with high sensitivity. As a demonstration, a novel colorimetric method based on Zr@PDA nanozyme was developed for sensitive detection of the drug fructose 1,6-diphosphate. The linear range is 1-15 µM with a detection limit as low as 0.38 µM.

`In crystallo' substrate binding triggers major domain movements and reveals magnesium as a co-activator of Trypanosoma brucei pyruvate kinase.

The active site of pyruvate kinase (PYK) is located between the AC core of the enzyme and a mobile lid corresponding to domain B. Many PYK structures have already been determined, but the first `effector-only' structure and the first with PEP (the true natural substrate) are now reported for the enzyme from Trypanosoma brucei. PEP soaked into crystals of the enzyme with bound allosteric activator fructose 2,6-bisphosphate (F26BP) and Mg(2+) triggers a substantial 23° rotation of the B domain `in crystallo', resulting in a partially closed active site. The interplay of side chains with Mg(2+) and PEP may explain the mechanism of the domain movement. Furthermore, it is apparent that when F26BP is present but PEP is absent Mg(2+) occupies a position that is distinct from the two canonical Mg(2+)-binding sites at the active site. This third site is adjacent to the active site and involves the same amino-acid side chains as in canonical site 1 but in altered orientations. Site 3 acts to sequester Mg(2+) in a `priming' position such that the enzyme is maintained in its R-state conformation. In this way, Mg(2+) cooperates with F26BP to ensure that the enzyme is in a conformation that has a high affinity for the substrate.

Sampling the conformational energy landscape of a hyperthermophilic protein by engineering key substitutions.

Proteins exist as a dynamic ensemble of interconverting substates, which defines their conformational energy landscapes. Recent work has indicated that mutations that shift the balance between conformational substates (CSs) are one of the main mechanisms by which proteins evolve new functions. In the present study, we probe this assertion by examining phenotypic protein adaptation to extreme conditions, using the allosteric tetrameric lactate dehydrogenase (LDH) from the hyperthermophilic bacterium Thermus thermophilus (Tt) as a model enzyme. In the presence of fructose 1, 6 bis-phosphate (FBP), allosteric LDHs catalyze the conversion of pyruvate to lactate with concomitant oxidation of nicotinamide adenine dinucleotide, reduced form (NADH). The catalysis involves a structural transition between a low-affinity inactive "T-state" and a high-affinity active "R-state" with bound FBP. During this structural transition, two important residues undergo changes in their side chain conformations. These are R171 and H188, which are involved in substrate and FBP binding, respectively. We designed two mutants of Tt-LDH with one ("1-Mut") and five ("5-Mut") mutations distant from the active site and characterized their catalytic, dynamical, and structural properties. In 1-Mut Tt-LDH, without FBP, the K(m)(Pyr) is reduced compared with that of the wild type, which is consistent with a complete shifting of the CS equilibrium of H188 to that observed in the R-state. By contrast, the CS populations of R171, k(cat) and protein stability are little changed. In 5-Mut Tt-LDH, without FBP, K(m)(Pyr) approaches the values it has with FBP and becomes almost temperature independent, k(cat) increases substantially, and the CS populations of R171 shift toward those of the R-state. These changes are accompanied by a decrease in protein stability at higher temperature, which is consistent with an increased flexibility at lower temperature. Together, these results show that the thermal properties of an enzyme can be strongly modified by only a few or even a single mutation, which serve to alter the equilibrium and, hence, the relative populations of functionally important native-state CSs, without changing the nature of the CSs themselves. They also provide insights into the types of mutational pathways by which protein adaptation to temperature is achieved.

Glycolytic and non-glycolytic functions of Mycobacterium tuberculosis fructose-1,6-bisphosphate aldolase, an essential enzyme produced by replicating and non-replicating bacilli.

The search for antituberculosis drugs active against persistent bacilli has led to our interest in metallodependent class II fructose-1,6-bisphosphate aldolase (FBA-tb), a key enzyme of gluconeogenesis absent from mammalian cells. Knock-out experiments at the fba-tb locus indicated that this gene is required for the growth of Mycobacterium tuberculosis on gluconeogenetic substrates and in glucose-containing medium. Surface labeling and enzymatic activity measurements revealed that this enzyme was exported to the cell surface of M. tuberculosis and produced under various axenic growth conditions including oxygen depletion and hence by non-replicating bacilli. Importantly, FBA-tb was also produced in vivo in the lungs of infected guinea pigs and mice. FBA-tb bound human plasmin(ogen) and protected FBA-tb-bound plasmin from regulation by α(2)-antiplasmin, suggestive of an involvement of this enzyme in host/pathogen interactions. The crystal structures of FBA-tb in the native form and in complex with a hydroxamate substrate analog were determined to 2.35- and 1.9-Å resolution, respectively. Whereas inhibitor attachment had no effect on the plasminogen binding activity of FBA-tb, it competed with the natural substrate of the enzyme, fructose 1,6-bisphosphate, and substantiated a previously unknown reaction mechanism associated with metallodependent aldolases involving recruitment of the catalytic zinc ion by the substrate upon active site binding. Altogether, our results highlight the potential of FBA-tb as a novel therapeutic target against both replicating and non-replicating bacilli.

Crystallization and preliminary X-ray characterization of the glpX-encoded class II fructose-1,6-bisphosphatase from Mycobacterium tuberculosis.

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), which is a key enzyme in gluconeogenesis, catalyzes the hydrolysis of fructose 1,6-bisphosphate to form fructose 6-phosphate and orthophosphate. The present investigation reports the crystallization and preliminary crystallographic studies of the glpX-encoded class II FBPase from Mycobacterium tuberculosis H37Rv. The recombinant protein, which was cloned using an Escherichia coli expression system, was purified and crystallized using the hanging-drop vapor-diffusion method. The crystals diffracted to a resolution of 2.7 Šand belonged to the hexagonal space group P6(1)22, with unit-cell parameters a = b = 131.3, c = 143.2 Å. The structure has been solved by molecular replacement and is currently undergoing refinement.

Structure of fructose bisphosphate aldolase from Bartonella henselae bound to fructose 1,6-bisphosphate.

Fructose bisphosphate aldolase (FBPA) enzymes have been found in a broad range of eukaryotic and prokaryotic organisms. FBPA catalyses the cleavage of fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. The SSGCID has reported several FBPA structures from pathogenic sources, including the bacterium Brucella melitensis and the protozoan Babesia bovis. Bioinformatic analysis of the Bartonella henselae genome revealed an FBPA homolog. The B. henselae FBPA enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme crystallized in the apo form but failed to diffract; however, well diffracting crystals could be obtained by cocrystallization in the presence of the native substrate fructose 1,6-bisphosphate. A data set to 2.35 Å resolution was collected from a single crystal at 100 K. The crystal belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=72.39, b=127.71, c=157.63 Å. The structure was refined to a final free R factor of 22.2%. The structure shares the typical barrel tertiary structure and tetrameric quaternary structure reported for previous FBPA structures and exhibits the same Schiff base in the active site.

A synthetic RNA-based biosensor for fructose-1,6-bisphosphate that reports glycolytic flux.

RNA-based sensors for intracellular metabolites are a promising solution to the emerging issue of metabolic heterogeneity. However, their development, i.e., the conversion of an aptamer into an in vivo-functional intracellular metabolite sensor, still harbors challenges. Here, we accomplished this for the glycolytic flux-signaling metabolite, fructose-1,6-bisphosphate (FBP). Starting from in vitro selection of an aptamer, we constructed device libraries with a hammerhead ribozyme as actuator. Using high-throughput screening in yeast with fluorescence-activated cell sorting (FACS), next-generation sequencing, and genetic-environmental perturbations to modulate the intracellular FBP levels, we identified a sensor that generates ratiometric fluorescent readout. An abrogated response in sensor mutants and occurrence of two sensor conformations-revealed by RNA structural probing-indicated in vivo riboswitching activity. Microscopy showed that the sensor can differentiate cells with different glycolytic fluxes within yeast populations, opening research avenues into metabolic heterogeneity. We demonstrate the possibility to generate RNA-based sensors for intracellular metabolites for which no natural metabolite-binding RNA element exits.

Changes in the allosteric site of human liver pyruvate kinase upon activator binding include the breakage of an intersubunit cation-π bond.

Human liver pyruvate kinase (hLPYK) converts phosphoenolpyruvate to pyruvate in the final step of glycolysis. hLPYK is allosterically activated by fructose-1,6-bisphosphate (Fru-1,6-BP). The allosteric site, as defined by previous structural studies, is located in domain C between the phosphate-binding loop (residues 444-449) and the allosteric loop (residues 527-533). In this study, the X-ray crystal structures of four hLPYK variants were solved to make structural correlations with existing functional data. The variants are D499N, W527H, Δ529/S531G (called GGG here) and S531E. The results revealed a conformational toggle between the open and closed positions of the allosteric loop. In the absence of Fru-1,6-BP the open position is stabilized, in part, by a cation-π bond between Trp527 and Arg538' (from an adjacent monomer). In the S531E variant glutamate binds in place of the 6'-phosphate of Fru-1,6-BP in the allosteric site, leading to partial allosteric activation. Finally, the structure of the D499N mutant does not provide structural evidence for the previously observed allosteric activation of the D499N variant.

Distinctive regulatory properties of pyruvate kinase 1 from Aedes aegypti mosquitoes.

Female Aedes aegypti mosquitoes are vectors of arboviruses that cause diseases of public health significance. The discovery of new metabolic targets is crucial for improving mosquito control strategies. We recently demonstrated that glucose oxidation supports ammonia detoxification in A. aegypti. Pyruvate kinase (PK, EC 2.7.1.40) catalyzes the last step of the glycolytic pathway. In most organisms, one or more allosteric effectors control PK activity. However, the kinetic properties and structure of PK in mosquitoes have not been previously reported. In this study, two alternatively spliced mRNA variants (AaPK1 and AaPK2) that code for PKs were identified in the A. aegypti genome. The AaPK1 mRNA variant, which encodes a 529 amino acid protein with an estimated molecular weight of ∼57 kDa, was cloned. The protein was expressed in Escherichia coli and purified. The AaPK1 kinetic properties were identified. The recombinant protein was also crystallized and its 3D structure determined. We found that alanine, glutamine, proline, serine and fructose-1-phosphate displayed a classic allosteric activation on AaPK1. Ribulose-5-phosphate acted as an allosteric inhibitor of AaPK1 but its inhibitory effect was reversed by alanine, glutamine, proline and serine. Additionally, the allosteric activation of AaPK1 by amino acids was weakened by fructose-1,6-bisphosphate, whereas the allosteric activation of AaPK1 by alanine and serine was diminished by glucose-6-phosphate. The AaPK1 structure shows the presence of fructose-1,6-bisphosphate in the allosteric site. Together, our results reveal that specific amino acids and phosphorylated sugars tightly regulate conformational dynamics and catalytic changes of AaPK1. The distinctive AaPK1 allosteric properties support a complex role for this enzyme within mosquito metabolism.

Structural mechanism for the fine-tuning of CcpA function by the small molecule effectors glucose 6-phosphate and fructose 1,6-bisphosphate.

In Gram-positive bacteria, carbon catabolite regulation (CCR) is mediated by the carbon catabolite control protein A (CcpA), a member of the LacI-GalR family of transcription regulators. Unlike other LacI-GalR proteins, CcpA is activated to bind DNA by binding the phosphoproteins HPr-Ser46-P or Crh-Ser46-P. However, fine regulation of CCR is accomplished by the small molecule effectors, glucose 6-phosphate (G6P) and fructose 1,6-bisphosphate (FBP), which somehow enhance CcpA-(HPr-Ser46-P) binding to DNA. Unlike the CcpA-(HPr-Ser46-P) complex, DNA binding by CcpA-(Crh-Ser46-P) is not stimulated by G6P or FBP. To understand the fine-tuning mechanism of these effectors, we solved the structures of the CcpA core, DeltaCcpA, which lacks the N-terminal DNA-binding domain, in complex with HPr-Ser46-P and G6P or FBP. G6P and FBP bind in a deep cleft, between the N and C subdomains of CcpA. Neither interacts with HPr-Ser46-P. This suggests that one role of the adjunct corepressors is to buttress the DNA-binding conformation effected by the binding of HPr-Ser46-P to the CcpA dimer N subdomains. However, the structures reveal that an unexpected function of adjunct corepressor binding is to bolster cross interactions between HPr-Ser46-P residue Arg17 and residues Asp69 and Asp99 of the other CcpA subunit. These cross contacts, which are weak or not present in the CcpA-(Crh-Ser46-P) complex, stimulate the CcpA-(HPr-Ser46-P)-DNA interaction specifically. Thus, stabilization of the closed conformation and bolstering of cross contacts between CcpA and its other corepressor, HPr-Ser46-P, provide a molecular explanation for how adjunct corepressors G6P and FBP enhance the interaction between CcpA-(HPr-Ser46-P) and cognate DNA.

Fructose-2,6-bisphosphate counteracts guanidinium chloride-, thermal-, and ATP-induced dissociation of skeletal muscle key glycolytic enzyme 6-phosphofructo-1-kinase: A structural mechanism for PFK allosteric regulation.

Rabbit muscle 6-phosphofructo-1-kinase (PFK) is the key glycolytic enzyme being regulated by diverse molecules and signals. This enzyme may undergo a reversible dissociation from a fully active homotetramer to a quite inactive dimer. There are evidences that some positive and negative modulators of PFK, such as ADP and citrate, may interfere with the enzyme oligomeric structure shifting the tetramer-dimer equilibrium towards opposite orientations, where the negative modulators favor the dissociation of tetramers into dimers and vice versa. PFK is allosterically inhibited by ATP at its physiological range of concentration, an effect counteracted by fructose-2,6-bisphosphate (F2,6BP). However, the structural molecular mechanism by which ATP and F2,6BP regulate PFK is hitherto demonstrated. The present paper aimed at demonstrating that either the ATP-induced inhibition of PFK and the reversion of this inhibition by F2,6BP occur through the same molecular mechanism, i.e., the displacement of the oligomeric equilibrium of the enzyme. This conclusion is arrived assessing the effects of ATP and F2,6BP on PFK inactivation through two distinct ways to dissociate the enzyme: (a) upon incubation at 50 degrees C, or (b) incubating the enzyme with guanidinium hydrochloride (GdmCl). Our results reveal that temperature- and GdmCl-induced inactivation of PFK prove remarkably more effective in the presence 5mM ATP than in the absence of additives. On the other hand, the presence of 100 nM F2,6BP attenuate the effects of both high-temperature exposition and GdmCl on PFK, even in the simultaneous presence of 5mM ATP. These data support the hypothesis that ATP shifts the oligomeric equilibrium of PFK towards the smaller conformations, while F2,6BP acts in the opposite direction. This conclusion leads to important information about the molecular mechanism by which PFK is regulated by these modulators.