Structural and enzymatic analysis of soybean beta-amylase mutants with increased pH optimum

Comparison of the architecture around the active site of soybean beta-amylase and Bacillus cereus beta-amylase showed that the hydrogen bond networks (Glu380-(Lys295-Met51) and Glu380-Asn340-Glu178) in soybean beta-amylase around the base catalytic residue, Glu380, seem to contribute to the lower pH optimum of soybean beta-amylase. To convert the pH optimum of soybean beta-amylase (pH 5.4) to that of the bacterial type enzyme (pH 6.7), three mutants of soybean beta-amylase, M51T, E178Y, and N340T, were constructed such that the hydrogen bond networks were removed by site-directed mutagenesis. The kinetic analysis showed that the pH optimum of all mutants shifted dramatically to a neutral pH (range, from 5.4 to 6.0-6.6). The Km values of the mutants were almost the same as that of soybean beta-amylase except in the case of M51T, while the Vmax values of all mutants were low compared with that of soybean beta-amylase. The crystal structure analysis of the wild type-maltose and mutant-maltose complexes showed that the direct hydrogen bond between Glu380 and Asn340 was completely disrupted in the mutants M51T, E178Y, and N340T. In the case of M51T, the hydrogen bond between Glu380 and Lys295 was also disrupted. These results indicated that the reduced pKa value of Glu380 is stabilized by the hydrogen bond network and is responsible for the lower pH optimum of soybean beta-amylase compared with that of the bacterial beta-amylase.     

Circular dichroism and the conformational properties of soybean beta-amylase

The conformational properties of soybean β-amylase were investigated by the circular dichroism probe and measurement of enzyme activity. The enzyme exhibited a positive circular dichroism band at 192 nm, a negative band at 222 nm, and a shoulder near 210 nm. Analysis of the spectrum in the far ultraviolet zone indicated the presence of approximately 30% of α helix and 5–10% of β-pleated sheet, the rest of the polypeptide main chain possessing aperiodic structure. In the near ultraviolet reagion, the enzyme protein showed at least six positive peaks at 259, 265, 273, 281, 292, and 297 nm. The positive bands at 292 and 297 nm remained unaltered on acetylation of the enzyme by N-acetylimidazole and were assigned to tryptophanyl chromophores. These bands were affected in intensity in the presence of maltose or cycloheptaamylose, which indicates that some tryptophan residues are situated at the binding sites. The native conformation of soybean β-amylase was found to be sensitive to pH variation (below pH 5 and above pH 10), sodium dodecyl sulfate, guanidine hydrochloride, and heating to 50–55 °C. Complete disorganization of the secondary structure was attained by 6 m guanidine hydrochloride. Sodium dodecyl sulfate was effective in disturbing the tertiary structure of the enzyme but did not affect significantly the secondary structure. Enzymatic inactivation was paralleled by the decrease of circular dichroism bands in the near ultraviolet region as produced by the denaturants. It is concluded that the uniquely folded structure of the enzyme contains some less rigid domains and a rigid core stabilized by hydrophobic interactions, electrostatic interactions, and hydrogen bonds.     

Large-scale conformational dynamics of the HIV-1 integrase core domain and its catalytic loop mutants

HIV-1 integrase is one of the three essential enzymes required for viral replication and has great potential as a novel target for anti-HIV drugs. Although tremendous efforts have been devoted to understanding this protein, the conformation of the catalytic core domain around the active site, particularly the catalytic loop overhanging the active site, is still not well characterized by experimental methods due to its high degree of flexibility. Recent studies have suggested that this conformational dynamics is directly correlated with enzymatic activity, but the details of this dynamics is not known. In this study, we conducted a series of extended-time molecular dynamics simulations and locally enhanced sampling simulations of the wild-type and three loop hinge mutants to investigate the conformational dynamics of the core domain. A combined total of >480 ns of simulation data was collected which allowed us to study the conformational changes that were not possible to observe in the previously reported short-time molecular dynamics simulations. Among the main findings are a major conformational change (>20 A) in the catalytic loop, which revealed a gatinglike dynamics, and a transient intraloop structure, which provided a rationale for the mutational effects of several residues on the loop including Q(148), P(145), and Y(143). Further, clustering analyses have identified seven major conformational states of the wild-type catalytic loop. Their implications for catalytic function and ligand interaction are discussed. The findings reported here provide a detailed view of the active site conformational dynamics and should be useful for structure-based inhibitor design for integrase.     

Backbone dynamics in dihydrofolate reductase complexes: role of loop flexibility in the catalytic mechanism

To elucidate the influence of local motion of the polypeptide chain on the catalytic mechanism of an enzyme, we have measured (15)N relaxation data for Escherichia coli dihydrofolate reductase in three different complexes, representing different stages in the catalytic cycle of the enzyme. NMR relaxation data were analyzed by the model-free approach, corrected for rotational anisotropy, to provide insights into the backbone dynamics. There are significant differences in the backbone dynamics in the different complexes. Complexes in which the cofactor binding site is occluded by the Met20 loop display large amplitude motions on the picosecond/nanosecond time scale for residues in the Met20 loop, the adjacent betaF-betaG loop and for residues 67-69 in the adenosine binding loop. Formation of the closed Met20 loop conformation in the ternary complex with folate and NADP(+), results in attenuation of the motions in the Met20 loop and the betaF-betaG loop but leads to increased flexibility in the adenosine binding loop. New fluctuations on a microsecond/millisecond time scale are observed in the closed E:folate:NADP(+) complex in regions that form hydrogen bonds between the Met20 and the betaF-betaG loops. The data provide insights into the changes in backbone dynamics during the catalytic cycle and point to an important role of the Met20 and betaF-betaG loops in controlling access to the active site. The high flexibility of these loops in the occluded conformation is expected to promote tetrahydrofolate-assisted product release and facilitate binding of the nicotinamide ring to form the Michaelis complex. The backbone fluctuations in the Met20 loop become attenuated once it closes over the active site, thereby stabilizing the nicotinamide ring in a geometry conducive to hydride transfer. Finally, the relaxation data provide evidence for long-range motional coupling between the adenosine binding loop and distant regions of the protein.     

Change in the crystal packing of soybean beta-amylase mutants substituted at a few surface amino acid residues

In spite of the high similarity of amino acid sequence and three-dimensional structure between soybean beta-amylase (SBA) and sweet potato beta-amylase (SPB), their quaternary structure is quite different, being a monomer in SBA and a tetramer in SPB. Because most of the differences in amino acid sequences are found in the surface region, we tested the tetramerization of SBA by examining mutations of residues located at the surface. We designed the SBA tetramer using the SPB tetramer structure as a model and calculating the change of accessible surface area (DeltaASA) for each residue in order to select sites for the mutation. Two different mutant genes encoding SB3 (D374Y/L481R/P487D) and SB4 (K462S added to SB3), were constructed for expression in Escherichia coli and the recombinant proteins were purified. They existed as a monomer in solution, but gave completely different crystals from the native SBA. The asymmetric unit of the mutants contains four molecules, while that of native SBA contains one. The interactions of the created interfaces revealed that there were more intermolecular interactions in the SB3 than in the SB4 tetramer. The substituted charged residues on the surface are involved in interactions with adjacent molecules in a different way, forming a new crystal packing pattern.     

The catalytic and conformational cycle of Aquifex aeolicus KDO8P synthase: role of the L7 loop

KDO8P synthase catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form the 8-carbon sugar KDO8P and inorganic phosphate (P(i)). The X-ray structure of the wild-type enzyme shows that when both PEP and A5P bind, the active site becomes isolated from the environment due to a conformational change of the L7 loop. The structures of the R106G mutant, without substrates, and with PEP and PEP plus A5P bound, were determined and reveal that in R106G closure of the L7 loop is impaired. The structural perturbations originating from the loss of the Arg(106) side chain point to a role of the L2 loop in stabilizing the closed conformation of the L7 loop. Despite the increased exposure of the R106G active site, no abnormal reaction of PEP with water was observed, ruling out the hypothesis that the primary function of the L7 loop is to shield the active site from bulk solvent during the condensation reaction. However, the R106G enzyme displays several kinetic abnormalities on both the substrate side (smaller K(m)(PEP), larger K(i)(A5P) and K(m)(A5P)) and the product side (smaller K(i)(Pi) and K(i)(KDO8P)) of the reaction. As a consequence, the mutant enzyme is less severely inhibited by A5P and more severely inhibited by P(i) and KDO8P. Simulations of the flux of KDO8P synthesis under metabolic steady-state conditions (constant concentration of reactants and products over time) suggest that in vivo R106G is expected to perform optimally in a narrower range of substrate and product concentrations than the wild-type enzyme.     

Crystal structures of threonine synthase from Thermus thermophilus HB8: conformational change,substrate recognition,and mechanism

Threonine synthase, which is a PLP-dependent enzyme, catalyzes the beta,gamma-replacement reaction of l-homoserine phosphate to yield threonine and inorganic phosphate. The three-dimensional structures of the enzyme from Thermus thermophilus HB8 in its unliganded form and complexed with the substrate analogue 2-amino-5-phosphonopentanoic acid have been determined at 2.15 and 2.0 A resolution, respectively. The complexed form, assigned as an enamine, uncovered the interactions of the cofactor-analogue conjugate with the active site residues. The binding of the substrate analogue induces a large conformational change at the domain level. The small domain rotates by about 25 degrees and approaches the large domain to close the active site. The complicated catalytic process of the enzyme has been elucidated based on the complex structure to reveal the stereochemistry of the reaction and to present the released inorganic phosphate as a possible catalyst to carry a proton to the Cgamma atom of the substrate.     

Structural basis for conformational equilibrium of the catalytic spliceosome

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Zinc-dependent cleavage in the catalytic core of the hammerhead ribozyme: evidence for a pH-dependent conformational change

We have characterized a novel Zn²⁺-catalyzed cleavage site between nucleotides C3 and U4 in the catalytic core of the hammerhead ribozyme. In contrast to previously described divalent metal-ion-dependent cleavage of RNA, U4 cleavage is only observed in the presence of Zn²⁺. This new cleavage site has an unusual pH dependence, in that U4 cleavage products are only observed above pH 7.9 and reach a maximum yield at about pH 8.5. These data, together with the fact that no metal ion-binding site is observed in proximity to the U4 cleavage site in either of the crystal structures, point toward a pH-dependent conformational change in the hammerhead ribozyme. We have described previously Zn²⁺-dependent cleavage between G8 and A9 in the hammerhead ribozyme and have discovered that U4 cleavage occurs only after A9 cleavage. To our knowledge, this is the first example of sequential cleavage events as a possible regulatory mechanism in ribozymes.     

Prediction of a ligand-induced conformational change in the catalytic core of Cdc25A

The cell cycle control phosphatases Cdc25 are dual specificity phosphatases that dephosphorylate both phosphothreonine and phosphotyrosine residues on their substrate proteins. The determination of the apo-protein structure of Cdc25A revealed that this enzyme has a completely different fold compared to all other phosphatases crystallised to date. The conformation of the active site residues does not seem very suitable for catalysis in this unliganded structure. We have studied some structural features of the Cdc25A apo-structure and a modelled Cdc25A-ligand complex by molecular dynamics simulations. The simulations predict a conformational change in the peptide backbone of the complex, which is not observed in the apo-structure. This ligand-induced conformational change yields a structure that is similar to other protein tyrosine phosphatase-ligand complexes that have been crystallised. The change in conformation takes place in the position between a serine and a glutamic acid residue in the phosphate binding loop. We suggest that this type of conformational change is an important molecular switch in the catalytic process.     

Structural analysis of the catalytic mechanism and stereoselectivity in Streptomyces coelicolor alditol oxidase

Alditol oxidase (AldO) from Streptomyces coelicolor A3(2) is a soluble monomeric flavin-dependent oxidase that performs selective oxidation of the terminal primary hydroxyl group of several alditols. Here, we report the crystal structure of the recombinant enzyme in its native state and in complex with both six-carbon (mannitol and sorbitol) and five-carbon substrates (xylitol). AldO shares the same folding topology of the members of the vanillyl-alcohol oxidase family of flavoenzymes and exhibits a covalently linked FAD which is located at the bottom of a funnel-shaped pocket that forms the active site. The high resolution of the three-dimensional structures highlights a well-defined hydrogen-bonding network that tightly constrains the substrate in the productive conformation for catalysis. Substrate binding occurs through a lock-and-key mechanism and does not induce conformational changes with respect to the ligand-free protein. A network of charged residues is proposed to favor catalysis through stabilization of the deprotonated form of the substrate. A His side chain acts as back door that "pushes" the substrate-reactive carbon atom toward the N5-C4a locus of the flavin. Analysis of the three-dimensional structure reveals possible pathways for diffusion of molecular oxygen and a small cavity on the re side of the flavin that may host oxygen during FAD reoxidation. These features combined with the tight shape of the catalytic site provide insights into the mechanism of AldO-mediated regioselective oxidation reactions and its substrate specificity.     

Sulfmyoglobin conformational change: A role in the decrease of oxy-myoglobin functionality

This work is focused at understanding the interaction of H₂S with Myoglobin (Mb), in particular the Sulfmyoglobin (SMb) product, whose physiological role is controversial and not well understood. The scattering curves, Guinier, Kratky, Porod and P(r) plots were analyzed for oxy-Mb and oxy-Hemoglobin I (oxyHbI) in the absence and presence of H₂S, using Small and Wide Angle X-ray Scattering (SAXS/WAXS) technique. Three dimensional models were also generated from the SAXS/WAXS data. The results show that SMb formation, produced by oxyMb and H₂S interaction, induces a change in the protein conformation where its envelope has a very small cleft and the protein is more flexible, less rigid and compact. Based on the direct relationship between Mb's structural conformation and its functionality, we suggest that the conformational change observed upon SMb formation plays a contribution to the protein decrease in O₂ affinity and, therefore, on its functionality.     

MgATP-induced conformational change of the catalytic subunit of cAMP-dependent protein kinase

Conformational changes of the cAMP-dependent protein kinase (PKA) catalytic (C) subunit are critical for the catalysis of gamma-phosphate transfer from adenosine 5'-triphosphate (ATP) to target proteins. Time-resolved fluorescence anisotropy (TRFA) was used to investigate the respective roles of Mg(2+), ATP, MgATP, and the inhibitor peptide (IP20) in the conformational changes of a 5,6-carboxyfluorescein succinimidyl ester (CF) labeled C subunit ((CF)C). TRFA decays were fit to a biexponential equation incorporating the fast and slow rotational correlation times phi(F) and phi(S). The (CF)C apoenzyme exhibited the rotational correlation times phi(F)=1.8+/-0.3 ns and phi(S)=20.1+/-0.6 ns which were reduced to phi(F)=1.1+/-0.2 ns and phi(S)=13.3+/-0.9 ns in the presence of MgATP. The reduction in rotational correlation times indicated that the (CF)C subunit adopted a more compact shape upon formation of a (CF)C.MgATP binary complex. Neither Mg(2+) (1-3 mM) nor ATP (0.4 mM) alone induced changes in the (CF)C subunit conformation equivalent to those induced by MgATP. The effect of MgATP was removed in the presence of ethylenediaminetetraacetic acid (EDTA). The addition of IP20 and MgATP to form the (CF)C x MgATP x IP20 ternary complex produced rotational correlation times similar to those of the (CF)C x MgATP binary complex. However, IP20 alone did not elicit an equivalent reduction in rotational correlation times. The results indicate that binding of MgATP to the C subunit may induce conformation changes in the C subunit necessary for the proper stereochemical alignment of substrates in the subsequent phosphorylation.     

Anatomy of a conformational transition of beta-strand 6 in soybean beta-amylase caused by substrate (or inhibitor) binding to the catalytical site.

A computational study of the five soybean beta-amylase X-ray structure reported so far revealed a peculiar conformational transition after substrate (or inhibitor) binding, which affects a segment of the beta-strand 6 (residues 341-343) in the (beta/alpha)8 molecular scaffold. Backbone distortions that involve considerable changes in the phi and psi angles were observed, as well as two sharp rotamer transitions for the Thr342 and Cys343 side chains. These changes caused the outermost CA-layer (at the C-terminal side of the barrel), which is involved in the catalysis, to shrink. Our observations strongly suggest that the 341FTC343 residue conformations in the free enzyme are not optimal for protein stability. Furthermore, as a result of conformational transitions in the ligand-binding process, there is a negative enthalpy change for these residues (-27 and -34 kcal/mol, after substrate or inhibitor binding, respectively). These findings support the proposed "stability-function" hypothesis for proteins that recognize a ligand (Shoichet BK, Baase WA, Kuroki R, Matthews BW. 1995. A relationship between protein stability and protein function. Proc Natl Acad Sci USA 92:452-456). They are also in good agreement with other experimental results in the literature that describe the role of the 341-343 segment in beta-amylase activity. Site-directed mutagenesis focused on these residues could be useful for undertaking functional studies of beta-amylase.     

A diffusion Michaelis-Menten mechanism: continuous conformational change in enzymatic kinetics

We present a simple model which extends the Michaelis-Menten mechanism by incorporating a continuous protein conformational change in enzymatic catalysis. This model can represent a quantitative version for "rack" or "induced fit" mechanisms. In the steady-state it leads to an equation of the Michaelis-Menten form, but with the catalytic step at the active site showing strong dependence on solvent viscosity. We suggest that a careful examination of solvent viscosity effects on enzymatic activity may serve as a test for the conformational change hypothesis.     

A picrotoxin-specific conformational change in the glycine receptor M2-M3 loop

The external loop linking the M2 and M3 transmembrane domains is crucial for coupling agonist binding to channel gating in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility scan previously showed that glycine activation increased the surface accessibility of 6 contiguous residues (Arg271-Lys276) toward the N-terminal end of the homomeric alpha1 GlyR M2-M3 loop. In the present study we used a similar approach to determine whether the allosteric antagonist, picrotoxin, could impose conformational changes to this domain that cannot be induced by varying agonist concentrations alone. Picrotoxin slowed the reaction rate of a sulfhydryl-containing compound (MTSET) with A272C, S273C, and L274C. Before interpreting this as a picrotoxin-specific conformational change, it was necessary to eliminate the possibility of steric competition between picrotoxin and MTSET. Accordingly, we showed that picrotoxin and the structurally unrelated blocker, bilobalide, were both trapped in the R271C GlyR in the closed state and that a point mutation to the pore-lining Thr6' residue abolished inhibition by both compounds. We also demonstrated that the picrotoxin dissociation rate was linearly related to the channel open probability. These observations constitute a strong case for picrotoxin binding in the pore. We thus conclude that the picrotoxin-specific effects on the M2-M3 loop are mediated allosterically. This suggests that the M2-M3 loop responds differently to the occupation of different binding sites.     

Structural and dynamic characterization of omega-conotoxin MVIIA: the binding loop exhibits slow conformational exchange

omega-Conotoxin MVIIA is a 25-residue, disulfide-bridged polypeptide from the venom of the sea snail Conus magus that binds to neuronal N-type calcium channels. It forms a compact folded structure, presenting a loop between Cys8 and Cys15 that contains a set of residues critical for its binding. The loop does not have a unique defined structure, nor is it intrinsically flexible. Broadening of a subset of resonances in the NMR spectrum at low temperature, anomalous temperature dependence of the chemical shifts of some resonances, and exchange contributions to J(0) from (13)C relaxation measurements reveal that conformational exchange affects the residues in this loop. The effects of this exchange on the calculated structure of omega-conotoxin MVIIA are discussed. The exchange appears to be associated with a change in the conformation of the disulfide bridge Cys8-Cys20. The implications for the use of the omega-conotoxins as a scaffold for carrying other functions is discussed.     

Structural analysis of a mutational hot-spot in the EcoRV restriction endonuclease: a catalytic role for a main chain carbonyl group.

Following random mutagenesis of the Eco RV endonuclease, a high proportion of the null mutants carry substitutions at Gln69. Such mutants display reduced rates for the DNA cleavage step in the reaction pathway, yet the crystal structures of wild-type Eco RV fail to explain why Gln69 is crucial for activity. In this study, crystal structures were determined for two mutants of Eco RV, with Leu or Glu at residue 69, bound to specific DNA. The structures of the mutants are similar to the native protein and no function can be ascribed to the side chain of the amino acid at this locus. Instead, the structures of the mutant proteins suggest that the catalytic defect is due to the positioning of the main chain carbonyl group. In the enzyme-substrate complex for Eco RV, the main chain carbonyl of Gln69 makes no interactions with catalytic functions but, in the enzyme-product complex, it coordinates a metal ion bound to the newly liberated 5'-phosphate. This re-positioning may be hindered in the mutant proteins. Molecular dynamics calculations indicate that the metal on the phosphoryl oxygen interacts with the carbonyl group upon forming the pentavalent intermediate during phosphodiester hydrolysis. A main chain carbonyl may thus play a role in catalysis by Eco RV.