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D Ringe

Publications and source records attributed to D Ringe.

At least 19 recordsLinked to original sources
PubMed2000

Methyl groups of thymine bases are important for nucleic acid recognition by DtxR.

The expression of diphtheria toxin is controlled by the diphtheria toxin repressor (DtxR). Under conditions of high iron concentration, DtxR binds the tox operator to inhibit transcription. To study how DNA binding specificity is achieved by this repressor, we solved the crystal structure of the nickel(II) activated DtxR(C102D) mutant complexed with a 43mer DNA duplex containing the DtxR consensus binding sequence. Structural analysis of this complex and comparison with a previously determined DtxR(C102D)-Ni(II)-tox operator ternary complex revealed unusual van der Waals interactions between Ser37/Pro39 of the repressor helix-turn-helix (HTH) motif and the methyl groups of specific thymine bases in the consensus binding sequence. Gel mobility shift assays utilizing deoxyuridine modified duplex DNA probes proved the importance of these interactions: the four methyl groups shown to interact with Ser37/Pro39 in the crystal structure contribute a total of 3.4 kcal/mol to binding energy. Thus, in addition to making base-specific hydrogen-bonding interactions to the DNA through its Gln43 residue, DtxR also recognizes methyl groups at certain positions in the DNA sequence with its Ser37 and Pro39 side chains, to achieve binding specificity toward its cognate operator sequences.

Amino Acid Sequence↗
PubMed2000

Crystal structure at 2.4 A resolution of Borrelia burgdorferi inosine 5'-monophosphate dehydrogenase: evidence of a substrate-induced hinged-lid motion by loop 6.

The conversion of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) is the committed and rate-limiting reaction in de novo guanine nucleotide biosynthesis. Inosine 5'- monophosphate dehydrogenase (IMPDH) is the enzyme that catalyzes the oxidation of IMP to XMP with the concomitant reduction of nicotinamide adenine dinucleotide (from NAD(+) to NADH). Because of its critical role in purine biosynthesis, IMPDH is a drug design target for anticancer, antiinfective, and immunosuppressive chemotherapy. We have determined the crystal structure of IMPDH from Borrelia burgdorferi, the bacterial spirochete that causes Lyme disease, with a sulfate ion bound in the IMP phosphate binding site. This is the first structure of IMPDH in the absence of substrate or cofactor where the active-site loop (loop 6), which contains the essential catalytic residue Cys 229, is clearly defined in the electron density. We report that a seven residue region of loop 6, including Cys229, is tilted more than 6 A away from its position in substrate- or substrate analogue-bound structures of IMPDH, suggestive of a conformational change. The location of this loop between beta6 and alpha6 links IMPDH to a family of beta/alpha barrel enzymes known to utilize this loop as a functional lid during catalysis. Least-squares minimization, root-mean-square deviation analysis, and inspection of the molecular surface of the loop 6 region in the substrate-free B. burgdorferi IMPDH and XMP-bound Chinese hamster IMPDH show that loop 6 follows a similar pattern of hinged rigid-body motion and indicates that IMPDH may be using loop 6 to bind and sequester substrate and to recruit an essential catalytic residue.

Amino Acid Sequence↗
PubMed2000

The catalytic pathway of cytochrome p450cam at atomic resolution.

Members of the cytochrome P450 superfamily catalyze the addition of molecular oxygen to nonactivated hydrocarbons at physiological temperature-a reaction that requires high temperature to proceed in the absence of a catalyst. Structures were obtained for three intermediates in the hydroxylation reaction of camphor by P450cam with trapping techniques and cryocrystallography. The structure of the ferrous dioxygen adduct of P450cam was determined with 0.91 angstrom wavelength x-rays; irradiation with 1.5 angstrom x-rays results in breakdown of the dioxygen molecule to an intermediate that would be consistent with an oxyferryl species. The structures show conformational changes in several important residues and reveal a network of bound water molecules that may provide the protons needed for the reaction.

Camphor↗
PubMed2000

Crystal structure of rabbit phosphoglucose isomerase, a glycolytic enzyme that moonlights as neuroleukin, autocrine motility factor, and differentiation mediator.

The multifunctional protein phosphoglucose isomerase, also known as neuroleukin, autocrine motility factor, and differentiation and maturation mediator, has different roles inside and outside the cell. In the cytoplasm, it catalyzes the second step in glycolysis. Outside the cell, it serves as a nerve growth factor and cytokine. We have determined the three-dimensional structure of rabbit muscle phosphoglucose isomerase complexed with the competitive inhibitor D-gluconate 6-phosphate by X-ray crystallography at 2.5 A resolution. The structure shows that the enzyme is a dimer with two alpha/beta-sandwich domains in each subunit. The location of the bound D-gluconate 6-phosphate inhibitor leads to the identification of residues involved in substrate specificity (Ser209, Ser159, Thr214, Thr217, and Thr211). The results of previously published kinetic studies suggest that a lysine and a histidine are involved in the catalytic mechanism. The crystal structure suggests active site residues Lys518 and His388 might be these residues. In addition, the positions of amino acid residues that are substituted in the genetic disease nonspherocytic hemolytic anemia suggest how these substitutions can result in altered catalysis or protein stability.

Amino Acid Sequence↗
PubMed2000

Observation of unstable species in enzyme-catalyzed transformations using protein crystallography.

Recent advances in rapid X-ray diffraction data collection methods, cryocrystallography, and other techniques have made it possible to visualize short-lived species in enzyme-catalyzed reactions directly at atomic resolution for a significant number of crystalline enzymes. The wide range of reaction types, intermediate lifetimes, and crystal characteristics means that different methods must be employed in each case, but there are enough examples now of successful structure determinations of normally unstable species to suggest guidelines for future investigations.

Animals↗
PubMed2000

Solvent mobility and the protein 'glass' transition.

Proteins and other biomolecules undergo a dynamic transition near 200 K to a glass-like solid state with small atomic fluctuations. This dynamic transition can inhibit biological function. To provide a deeper understanding of the relative importance of solvent mobility and the intrinsic protein energy surface in the transition, a novel molecular dynamics simulation procedure with the protein and solvent at different temperatures has been used. Solvent mobility is shown to be the dominant factor in determining the atomic fluctuations above 180 K, although intrinsic protein effects become important at lower temperatures. The simulations thus complement experimental studies by demonstrating the essential role of solvent in controlling functionally important protein fluctuations.

Computer Simulation↗
PubMed2000

The role of residues outside the active site: structural basis for function of C191 mutants of Escherichia coli aspartate aminotransferase.

In previous kinetic studies of Escherichia coli aspartate aminotransferase, it was determined that some substitutions of conserved cysteine 191, which is located outside of the active site, altered the kinetic parameters of the enzyme (Gloss,L.M., Spencer,D. E. and Kirsch,J.F., 1996, Protein Struct. Funct. Genet., 24, 195-208). The mutations resulted in an alkaline shift of 0.6-0.8 pH units for the pK(a) of the internal aldimine between the PLP cofactor and Lys258. The change in the pK(a) affected the pH dependence of the k(cat)/K(m) (aspartate) values for the mutant enzymes. To help to understand these observations, crystal structures of five mutant forms of E.coli aspartate aminotransferase (the maleate complexes of C191S, C191F, C191Y and C191W, and C191S without maleate) were determined at about 2 A resolution in the presence of the pyridoxal phosphate cofactor. The overall three-dimensional fold of each mutant enzyme is the same as that of the wild-type protein, but there is a rotation of the mutated side chain around its C(alpha)-C(beta) bond. This side chain rotation results in a change in the pattern of hydrogen bonding connecting the mutant residue and the protonated Schiff base of the cofactor, which could account for the altered pK(a) of the Schiff base imine nitrogen that was reported previously. These results demonstrate how residues outside the active site can be important in helping determine the subtleties of the active site amino acid geometries and interactions and how mutations outside the active site can have effects on catalysis. In addition, these results help explain the surprising result previously reported that, for some mutant proteins, replacement of a buried cysteine with an aromatic side chain did not destabilize the protein fold. Instead, rotation around the C(alpha)-C(beta) bond allowed each large aromatic side chain to become buried in a nearby pocket without large changes in the enzyme's backbone geometry.

Amino Acid Substitution↗
PubMed1999

Crystal structure of the proenzyme domain of plasminogen.

We have solved the X-ray crystal structure of the proenzyme form of the catalytic domain of plasminogen, with the nonessential mutations M585Q, V673M, and M788L, to 2.0 A resolution. The structure presents an inactive protease characterized by Asp740 (chymotrypsinogen 194) hydrogen bonded to His586 (chymotrypsinogen 40), preventing proper formation of the oxyanion hole and S1 specificity pocket. In addition, the catalytic triad residues are misplaced relative to the active conformation adopted by serine proteases in the chymotrypsin family. Finally, a unique form of zymogen inactivation is observed, characterized by a "foot-in-mouth" mechanism in which Trp761 (chymotrypsinogen 215) is folded into the S1 specificity pocket preventing substrate binding.

Amino Acid Sequence↗
PubMed1999

1-Butaneboronic acid binding to Aeromonas proteolytica aminopeptidase: a case of arrested development.

Hydrolases containing two metal ions connected by a bridging ligand catalyze reactions important in carcinogensis, tissue repair, post-translational modification, control and regulation of biochemical pathways, and protein degradation. The aminopeptidase from Aeromonas proteolytica serves as a paradigm for the study of such bridged bimetallic proteases since its three-dimensional structure is known to very high resolution and its catalytic reaction is amenable to spectroscopic examination. Herein, we report the X-ray crystal structure at 1.9 A resolution of AAP complexed with 1-butaneboronic acid (BuBA). This structure suggests that this complex represents a snapshot of the proteolytic reaction in an arrested form between the Michaelis complex and the transition state. Comparison of the structure with spectroscopic and other data allows us to conclude that the apparently structurally symmetrical dizinc site is actually asymmetric electrostatically.

Aeromonas↗
PubMed1999

Structure of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli, a key enzyme in the aspartate family of amino acid biosynthesis.

Aspartate beta-semialdehyde dehydrogenase (ASADH) lies at the first branch point in an essential aspartic biosynthetic pathway found in bacteria, fungi and the higher plants. Mutations in the asd gene encoding for ASADH that produce an inactive enzyme are lethal, which suggests that ASADH may be an effective target for antibacterial, herbicidal and fungicidal agents. We have solved the crystal structure of the Escherichia coli enzyme to 2.5 A resolution using single isomorphous replacement and 3-fold non-crystallographic symmetry. Each monomer has an N-terminal nucleotide-binding domain and a dimerisation domain. The presence of an essential cysteine locates the active site in a cleft between the two domains. The functional dimer has the appearance of a butterfly, with the NADP-binding domains forming the wings and the dimerisation domain forming the body.A histidine residue is identified as a likely acid/base catalyst in the enzymic reaction. Other amino acids implicated in the enzymic activity by mutagenesis are found in the active site region and define the substrate binding pocket.

Amino Acid Sequence↗
PubMed1999

The role of water in the catalytic efficiency of triosephosphate isomerase.

The structural basis for the effect of the S96P mutation in chicken triosephosphate isomerase (cTIM) has been analyzed using a combination of X-ray crystallography and Fourier transform infrared spectroscopy. The X-ray structure is that of the enzyme complexed with phosphoglycolohydroxamate (PGH), an intermediate analogue, solved at a resolution of 1.9 A. The S96P mutation was identified as a second-site reverent when catalytically crippled mutants, E165D and H95N, were subjected to random mutagenesis. The presence of the second mutation leads to enhanced activity over the single mutation. However, the effect of the S96P mutation alone is to decrease the catalytic efficiency of the enzyme. The crystal structures of the S96P double mutants show that this bulky proline side chain alters the water structure within the active-site cavity (E165D; ref 1) and prevents nonproductive binding conformations of the substrate (H95N; ref 2). Comparison of the S96P single mutant structure with those of the wild-type cTIM, those of the single mutants (E165D and H95N), and those of the double mutants (E165D/S96P and H95N/S96P) begins to address the role of the conserved serine residue at this position. The results indicate that the residue positions the catalytic base E165 optimally for polarization of the substrate carbonyl, thereby aiding in proton abstraction. In addition, this residue is involved in positioning critical water molecules, thereby affecting the way in which water structure influences activity.

Animals↗
PubMed1999

Structure of a Michaelis complex analogue: propionate binds in the substrate carboxylate site of alanine racemase.

The structure of alanine racemase from Bacillus stearothermophilus with the inhibitor propionate bound in the active site was determined by X-ray crystallography to a resolution of 1.9 A. The enzyme is a homodimer in solution and crystallizes with a dimer in the asymmetric unit. Both active sites contain a pyridoxal 5'-phosphate (PLP) molecule in aldimine linkage to Lys39 as a protonated Schiff base, and the pH-independence of UV-visible absorption spectra suggests that the protonated PLP-Lys39 Schiff base is the reactive form of the enzyme. The carboxylate group of propionate bound in the active site makes numerous interactions with active-site residues, defining the substrate binding site of the enzyme. The propionate-bound structure therefore approximates features of the Michaelis complex formed between alanine racemase and its amino acid substrate. The structure also provides evidence for the existence of a carbamate formed on the side-chain amino group of Lys129, stabilized by interactions with one of the residues interacting with the carboxylate group of propionate, Arg136. We propose that this novel interaction influences both substrate binding and catalysis by precisely positioning Arg136 and modulating its charge.

Alanine Racemase↗
PubMed1999

Effects of the E177K mutation in D-amino acid transaminase. Studies on an essential coenzyme anchoring group that contributes to stereochemical fidelity.

D-Amino acid transaminase is a bacterial enzyme that uses pyridoxal phosphate (PLP) as a cofactor to catalyze the conversion of D-amino acids into their corresponding alpha-keto acids. This enzyme has already been established as a target for novel antibacterial agents through suicide inactivation by a number of compounds. To improve their potency and specificity, the detailed enzyme mechanism, especially the role of its PLP cofactor, is under investigation. Many PLP-dependent transaminases have a negatively charged amino acid residue forming a salt-bridge with the pyridine nitrogen of its cofactor that promotes its protonation to stabilize the formation of a ketimine intermediate, which is subsequently hydrolyzed in the normal transaminase reaction pathway. However, alanine racemase has a positively charged arginine held rigidly in place by an extensive hydrogen bond network that may destabilize the ketimine intermediate, and make it too short-lived for a transaminase type of hydrolysis to occur. To test this hypothesis, we changed Glu-177 into a titratable, positively charged lysine (E177K). The crystal structure of this mutant shows that the positive charge of the newly introduced lysine side chain points away from the nitrogen of the cofactor, which may be due to electrostatic repulsions not being overcome by a hydrogen bond network such as found in alanine racemase. This mutation makes the active site more accessible, as exemplified by both biochemical and crystallographic data: CD measurements indicated a change in the microenvironment of the protein, some SH groups become more easily titratable, and at pH 9.0 the PMP peak appeared around 315 nm rather than at 330 nm. The ability of this mutant to convert L-alanine into D-alanine increased about 10-fold compared to wild-type and to about the same extent as found with other active site mutants. On the other hand, the specific activity of the E177K mutant decreased more than 1000-fold compared to wild-type. Furthermore, titration with L-alanine resulted in the appearance of an enzyme-substrate quinonoid intermediate absorbing around 500 nm, which is not observed with usual substrates or with the wild-type enzyme in the presence of L-alanine. The results overall indicate the importance of charged amino acid side chains relative to the coenzyme to maintain high catalytic efficiency.

Amino Acid Substitution↗
PubMed1999

Analysis of the binding surfaces of proteins.

We have developed an experimental approach to map the complete binding surface of any crystalline macromolecule that is fast and flexible. Crystals of the target protein are transferred into organic solvents and the crystal structures are determined at high (about 2A) resolution. The sites where the solvent molecules bind to the protein are thus identified directly. Different solvents serve as probes for different organic functional groups; thus, benzene is a probe for where aromatic groups like to bind, dimethyl formamide is a probe for peptide binding sites, and so forth. A series of about six such experiments suffices to locate the major binding regions on the protein surface unambiguously. These different sites can then be targeted with "Hydra-headed" inhibitors that interact simultaneously with more that one site, thereby providing specificity for the desired target. We have used this method to map the complete binding surface of elastase, and find that three regions, including the active site cleft, are generally "sticky" and can make interactions with almost any functional group. Analyses of these binding sites on elastase and other proteins suggests that what makes a binding site is amphipathicity and the ease with which water can be displaced.

Binding Sites↗
PubMed1999

Comparison of anionic and cationic trypsinogens: the anionic activation domain is more flexible in solution and differs in its mode of BPTI binding in the crystal structure.

Unlike bovine cationic trypsin, rat anionic trypsin retains activity at high pH. This alkaline stability has been attributed to stabilization of the salt bridge between the N-terminal Ile16 and Asp194 by the surface negative charge (Soman K, Yang A-S, Honig B, Fletterick R., 1989, Biochemistry 28:9918-9926). The formation of this salt bridge controls the conformation of the activation domain in trypsin. In this work we probe the structure of rat trypsinogen to determine the effects of the surface negative charge on the activation domain in the absence of the Ile16-Asp194 salt bridge. We determined the crystal structures of the rat trypsin-BPTI complex and the rat trypsinogen-BPTI complex at 1.8 and 2.2 A, respectively. The BPTI complex of rat trypsinogen resembles that of rat trypsin. Surprisingly, the side chain of Ile16 is found in a similar position in both the rat trypsin and trypsinogen complexes, although it is not the N-terminal residue and cannot form the salt bridge in trypsinogen. The resulting position of the activation peptide alters the conformation of the adjacent autolysis loop (residues 142-153). While bovine trypsinogen and trypsin have similar CD spectra, the CD spectrum of rat trypsinogen has only 60% of the intensity of rat trypsin. This lower intensity most likely results from increased flexibility around two conserved tryptophans, which are adjacent to the activation domain. The NMR spectrum of rat trypsinogen contains high field methyl signals as observed in bovine trypsinogen. It is concluded that the activation domain of rat trypsinogen is more flexible than that of bovine trypsinogen, but does not extend further into the protein core.

Amino Acid Sequence↗
PubMed1998

Evolution of an enzyme active site: the structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase.

Muconate lactonizing enzyme (MLE), a component of the beta-ketoadipate pathway of Pseudomonas putida, is a member of a family of related enzymes (the "enolase superfamily") that catalyze the abstraction of the alpha-proton of a carboxylic acid in the context of different overall reactions. New untwinned crystal forms of MLE were obtained, one of which diffracts to better than 2.0-A resolution. The packing of the octameric enzyme in this crystal form is unusual, because the asymmetric unit contains three subunits. The structure of MLE presented here contains no bound metal ion, but is very similar to a recently determined Mn2+-bound structure. Thus, absence of the metal ion does not perturb the structure of the active site. The structures of enolase, mandelate racemase, and MLE were superimposed. A comparison of metal ligands suggests that enolase may retain some characteristics of the ancestor of this enzyme family. Comparison of other residues involved in catalysis indicates two unusual patterns of conservation: (i) that the position of catalytic atoms remains constant, although the residues that contain them are located at different points in the protein fold; and (ii) that the positions of catalytic residues in the protein scaffold are conserved, whereas their identities and roles in catalysis vary.

Binding Sites↗