Biomedical subjects

G E Schulz

Publications and source records attributed to G E Schulz.

At least 73 records · Page 4Linked to original sources

PubMed1995

High-resolution structures of adenylate kinase from yeast ligated with inhibitor Ap5A, showing the pathway of phosphoryl transfer.

The structure of adenylate kinase from yeast ligated with the two-substrate-mimicking inhibitor Ap5A and Mg2+ has been refined to 1.96 A resolution. In addition, the refined structure of the same complex with a bound imidazole molecule replacing Mg2+ has been determined at 1.63 A. These structures indicate that replacing Mg2+ by imidazole disturbs the water structure and thus the complex. A comparison with the G-proteins shows that Mg2+ is exactly at the same position with respect to the phosphates. However, although the Mg2+ ligand sphere of the G-proteins is a regular octahedron containing peptide ligands, the reported adenylate kinase has no such ligands and an open octahedron leaving space for the Mg2+ to accompany the transferred phosphoryl group. A superposition of the known crystalline and therefore perturbed phosphoryl transfer geometries in the adenylate kinases demonstrates that all of them are close to the start of the forward reaction with bound ATP and AMP. Averaging all observed perturbed structures gives rise to a close approximation of the transition state, indicating in general how to establish an elusive transition state geometry. The average shows that the in-line phosphoryl transfer is associative, because there is no space for a dissociative metaphosphate intermediate. As a side result, the secondary dipole interaction in the alpha-helices of both protein structures has been quantified.

Adenosine Monophosphate↗

PubMed1994

Crystallization and X-ray crystallographic analysis of recombinant chicken poly(ADP-ribose) polymerase catalytic domain produced in Sf9 insect cells.

Poly (ADP-ribose) polymerase (PARP) participates in the immediate response in mammalian cells exposed to DNA-damaging agents. Recombinant baculovirus harboring the cDNA of the chicken PARP catalytic domain (40 kDa) have been used to infect Spodoptera frugiperda (Sf9) insect cells. The recombinant polypeptide (30 mg per 1 x 10(9) cells) was purified to homogeneity by 3-aminobenzamide affinity chromatography. The enzymatic properties of the recombinant domain were similar to those of the native fragment. Crystals of the purified recombinant catalytic domain were grown by vapor diffusion. The crystals belong to space group P2(1)2(1)2(1) with unit cell dimensions of a = 59.2 A, b = 65.0 A, c = 96.9 A. They are suitable for X-ray analysis and diffract to 2.0 A.

Animals↗

PubMed1994

Refined structure of the porin from Rhodopseudomonas blastica. Comparison with the porin from Rhodobacter capsulatus.

The structure of the membrane channel porin from the phototrophic bacteria Rhodopseudomonas blastica has been refined at 1.96 A resolution yielding an R-factor of 17.6%. The final model consists of all 289 amino acid residues, 247 water molecules and three detergent molecules modelled as n-octyltetraoxyethylene. One of these detergent molecules binds together with its two symmetry-related molecules tightly in a pocket at the molecular 3-fold axis. This pocket may bind three alkyl chains of a lipopolysaccharide which in turn would stabilize the trimer and could possibly play a role in membrane insertion. The overall shape of this porin resembles OmpF of Escherichia coli more than the only known sequence-related porin from Rhodobacter capsulatus. The membrane contacting surface is similar in all structurally known porins; it shows exceptional frequencies of amino acid residues and side-chain rotamers. The 46-residue loop beta 5-beta 6 of the porin is shown to be tightly fastened to the beta-barrel, excluding an in vivo loop movement that closes the pore. The trimer interface region has the structure of a water-soluble protein with an extensive non-polar core and numerous hydrogen bonds at the surface. The loops at the external end of the barrel are long and rigid whereas those at the periplasmic barrel end are short and mobile. The crystal packing is discussed.

Amino Acid Sequence↗

PubMed1994

The refined structures of a stabilized mutant and of wild-type pyruvate oxidase from Lactobacillus plantarum.

The crystal structure of pyruvate oxidase (EC 1.2.3.3) from Lactobacillus plantarum stabilized by three point mutations has been refined at 2.1 A resolution using the simulated annealing method. Based on 87,775 independent reflections in the resolution range 10 to 2.1 A, a final R-factor of 16.2% was obtained at good model geometry. The wild-type enzyme crystallizes isomorphously with the stabilized enzyme and has been analyzed at 2.5 A resolution. Pyruvate oxidase is a homotetramer with point group symmetry D2. One 2-fold axis is crystallographic, the others are local. The crystallographic asymmetric unit contains two subunits, and the model consists of the two polypeptide chains (residues 9 through 593), two FAD, two ThDP*Mg2+ and 739 water molecules. Each subunit has three domains; the CORE domain, the FAD domain and the ThDP domain. The FAD-binding chain fold is different from those of other known flavoproteins, whereas the ThDP-binding chain fold resembles the corresponding folds of the two other ThDP enzymes whose structure is known, transketolase and pyruvate decarboxylase. The peptide environment most likely forces the pyrimidine ring of ThDP into an unusual tautomeric form, which is required for catalysis. The structural differences between the wild-type and the stabilized enzyme are small. All three point mutations are at or near to the subunit interfaces, indicating that they stabilize the quarternary structure as had been deduced from reconstitution experiments.

Amino Acid Sequence↗

PubMed1994

The structure of uridylate kinase with its substrates, showing the transition state geometry.

Uridylate kinase from Saccharomyces cerevisiae is a member of the nucleoside monophosphate (NMP) kinase family and catalyzes the reaction ATP+NMP<==>ADP+NDP with moderate specificity for UMP. The recombinant enzyme crystallized together with two substrate molecules. The structure was solved, by multiple isomorphous replacement and solvent flattening, at 3.0 A and then refined at 2.13 A resolution. The present R-factor is 19%. Superposition onto the structure of a substrate-free adenylate kinase revealed the motions induced by substrate binding. A further superposition onto an adenylate kinase with bound P1,P5-bis(5'-adenosyl)pentaphosphate (Ap5A), a two-substrate-mimicking inhibitor, failed to explain the UMP preference of the uridylate kinase, but superimposed the nucleosides and in particular the non-transferred phosphates at the ATP- and NMP-site rather well. The coincidence of the phosphates indicate strongly that these groups assume their final positions during catalysis. This locates the transition state, which can be modeled with reasonable geometry in agreement with an in-line associative SN2 mechanism.

Adenosine Diphosphate↗

PubMed1994

Structure of the membrane channel porin from Rhodopseudomonas blastica at 2.0 A resolution.

The crystal structure of a membrane channel, homotrimeric porin from Rhodopseudomonas blastica has been determined at 2.0 A resolution by multiple isomorphous replacement and structural refinement. The current model has an R-factor of 16.5% and consists of 289 amino acids, 238 water molecules, and 3 detergent molecules per subunit. The partial protein sequence and subsequently the complete DNA sequence were determined. The general architecture is similar to those of the structurally known porins. As a particular feature there are 3 adjacent binding sites for n-alkyl chains at the molecular 3-fold axis. The side chain arrangement in the channel indicates a transverse electric field across each of the 3 pore eyelets, which may explain the discrimination against nonpolar solutes. Moreover, there are 2 significantly ordered girdles of aromatic residues at the nonpolar/polar borderlines of the interface between protein and membrane. Possibly, these residues shield the polypeptide conformation against adverse membrane fluctuations.

Amino Acid Sequence↗

PubMed1994

Structure of glutathione reductase from Escherichia coli at 1.86 A resolution: comparison with the enzyme from human erythrocytes.

The crystal structure of the dimeric flavoenzyme glutathione reductase from Escherichia coli was determined and refined to an R-factor of 16.8% at 1.86 A resolution. The molecular 2-fold axis of the dimer is local but very close to a possible crystallographic 2-fold axis; the slight asymmetry could be rationalized from the packing contacts. The 2 crystallographically independent subunits of the dimer are virtually identical, yielding no structural clue on possible cooperativity. The structure was compared with the well-known structure of the homologous enzyme from human erythrocytes with 52% sequence identity. Significant differences were found at the dimer interface, where the human enzyme has a disulfide bridge, whereas the E. coli enzyme has an antiparallel beta-sheet connecting the subunits. The differences at the glutathione binding site and in particular a deformation caused by a Leu-Ile exchange indicate why the E. coli enzyme accepts trypanothione much better than the human enzyme. The reported structure provides a frame for explaining numerous published engineering results in detail and for guiding further ones.

Amino Acid Sequence↗

PubMed1994

Anatomy of an engineered NAD-binding site.

The coenzyme specificity of Escherichia coli glutathione reductase was switched from NADP to NAD by modifying the environment of the 2'-phosphate binding site through a set of point mutations: A179G, A183G, V197E, R198M, K199F, H200D, and R204P (Scrutton NS, Berry A, Perham RN, 1990, Nature 343:38-43). In order to analyze the structural changes involved, we have determined 4 high-resolution crystal structures, i.e., the structures of the wild-type enzyme (1.86 A resolution, R-factor of 16.8%), of the wild-type enzyme ligated with NADP (2.0 A, 20.8%), of the NAD-dependent mutant (1.74 A, 16.8%), and of the NAD-dependent mutant ligated with NAD (2.2 A, 16.9%). A comparison of these structures reveals subtle differences that explain details of the specificity change. In particular, a peptide rotation occurs close to the adenosine ribose, with a concomitant change of the ribose pucker. The mutations cause a contraction of the local chain fold. Furthermore, the engineered NAD-binding site assumes a less rigid structure than the NADP site of the wild-type enzyme. A superposition of the ligated structures shows a displacement of NAD versus NADP such that the electron pathway from the nicotinamide ring to FAD is elongated, which may explain the lower catalytic efficiency of the mutant. Because the nicotinamide is as much as 15 A from the sites of the mutations, this observation reminds us that mutations may have important long-range consequences that are difficult to anticipate.

Binding Sites↗

PubMed1993

A thiamin diphosphate binding fold revealed by comparison of the crystal structures of transketolase, pyruvate oxidase and pyruvate decarboxylase.

BACKGROUND: The crystal structures of three thiamin diphosphate-dependent enzymes that catalyze distinct reactions in basic metabolic pathways are known. These enzymes--transketolase, pyruvate oxidase and pyruvate decarboxylase--also require metal ions such as Ca2+ and Mg2+ as cofactors and have little overall sequence similarity. Here, the crystal structures of these three enzymes are compared. RESULTS: The three enzymes share a similar pattern of binding of thiamin diphosphate and the metal ion cofactors. The enzymes function as multisubunit proteins, with each polypeptide chain folded into three alpha/beta domains. Two of these domains are involved in binding of the thiamin diphosphate and the metal ion. These domains have the same topology of six parallel beta-strands and surrounding alpha-helices. The thiamin diphosphate is bound in a cleft, formed by two domains from two different subunits. Only a few residues are conserved in all three enzymes and these are responsible for proper binding of the cofactors. CONCLUSIONS: Despite considerable differences in quaternary structure and lack of overall sequence homology, thiamin diphosphate binds to the three enzymes in a very similar fashion, and a general thiamin-binding fold can be revealed.

Amino Acid Sequence↗

PubMed1993

The spatial structure of the class II L-fuculose-1-phosphate aldolase from Escherichia coli.

The three-dimensional structure of L-fuculose-1-phosphate aldolase (FucA) from Escherichia coli was determined by X-ray crystallography at a resolution of 2.13 A. The enzyme is a homotetramer with an M(r) of 23,775 per subunit. Since its activity depends on the presence of metal ions (Zn2+) the enzyme belongs to the class II aldolases. As expected from amino acid sequence comparisons, this first structure of a class II aldolase shows no similarity to the known structures of class I aldolases. It has some unusual features concerning the overall chain fold, the quaternary structure, and the co-ordination of the catalytically active zinc ion. A sequence comparison with the data bank indicated that the middle domain of the enzyme L-ribulose-5-phosphate-4-epimerase is homologous to FucA and may contain an active-center metal ion.

Aldehyde-Lyases↗

PubMed1993

Porin conformation in the absence of calcium. Refined structure at 2.5 A resolution.

The crystal structure of porin from Rhodobacter capsulatus in the absence of divalent calcium ions has been refined to convergence at a resolution of 2.5 A using the simulated annealing refinement method. The final model consists of all 301 amino acid residues, 77 solvent molecules, one tris(hydroxymethyl)-aminomethane molecule and one unknown ligand modeled as n-octyltetraoxyethylene. A superposition with the previously described model containing three calcium ions showed structural changes at the segment 108-116 of the inner loop beta 5-beta 6, and at loops beta 8-beta 9 and beta 11-beta 12 at the extracellular side of the porin molecule. Evidence is presented that the conformational changes depend on the presence or absence of calcium ions. A possible influence on porin function is discussed.

Amino Acid Sequence↗

PubMed1993

Structural differences between wild-type NADP-dependent glutathione reductase from Escherichia coli and a redesigned NAD-dependent mutant.

NAD and NADP are ubiquitous coenzymes in biological redox reactions. They have distinct metabolic functions, yet they differ only by an additional phosphate group esterified at the 2'-hydroxyl group of the AMP moiety of NADP. The natural specificity of Escherichia coli glutathione reductase for NADP has previously been converted into a marked preference for NAD by introducing seven point mutations into the beta alpha beta-fold of the NADP-binding domain of the protein based on the known structure of the human enzyme. Among them was the replacement of Ala179 by glycine (A179G) in the alpha-helix of the fold, a change suggested by a difference in a sequence fingerprint previously found in the dinucleotide-binding domains of a number of dehydrogenases. Although this position is at a distance of 10 A from the bound 2'-phosphate group of NADP in glutathione reductase, the A179G mutation was found to be synergistic and beneficial. We have now carried out X-ray crystallographic analyses of the NAD-dependent mutant without and with bound NADH. A comparison of the structures of the mutant and wild-type enzymes reveals a flip of the peptide bond between Gly174 and Ala175 such that the side-chain of another introduced amino acid, Glu197, is fixed and can participate in binding the adenine ribose of NAD, thereby contributing to the ability of the mutated enzyme to exert its selectivity for the "wrong" coenzyme.

Amino Acid Sequence↗

PubMed1993

Purification and sequence determination of guanylate kinase from pig brain.

A purification procedure for guanylate kinase from pig brain has been developed consisting of ammonium sulfate precipitation and heptane extraction of the crude extract, hydrophobic-interaction chromatography, affinity chromatography and chromatofocussing. From 1.75 kg pig brain, 1.2 mg enzyme was isolated with a yield of 18% and a purity of about 90%. For sequence determination, the protein was cleaved with trypsin, cyanogen bromide and endoproteinase Glu-C. Some of the isolated peptides were subcleaved with chymotrypsin, thermolysin or trifluoroacetic acid. The blocked N-terminus was analyzed by mass spectrometry and by amino acid analysis of a tryptic peptide, while the C-terminus was found in a tryptic and a chymotryptic peptide and confirmed by a carboxypeptidase Y digestion. The sequence contains 197 amino acids with a M(r) of 21,831, one tryptophan and one cysteine residue. It has been compared to those of the homologous enzymes of yeast and Escherichia coli, as well as to proteins from sequence data banks that show similarities. The sequence is discussed in the light of the known spatial structure of yeast guanylate kinase.

Amino Acid Sequence↗

PubMed1993

Structure of the thiamine- and flavin-dependent enzyme pyruvate oxidase.

Pyruvate oxidase from Lactobacillus plantarum is a tetrameric enzyme that decarboxylates pyruvate, producing hydrogen peroxide and the energy-storage metabolite acetylphosphate. Structure determination at 2.1 angstroms showed that the cofactors thiamine pyrophosphate (TPP) and flavin adenine dinucleotide (FAD) are bound at the carboxyl termini of six-stranded parallel beta sheets. The pyrophosphate moiety of TPP is bound to a metal ion and to a beta alpha alpha beta unit corresponding to an established sequence fingerprint. The spatial arrangement of TPP and FAD suggests that the oxidation of the oxyethyl intermediate does not occur by hydride displacement but rather by a two-step transfer of two electrons.

Amino Acid Sequence↗

PubMed1993

NADH binding site and catalysis of NADH peroxidase.

The structure of the complex between cofactor NADH and the enzyme NADH peroxidase from Streptococcus faecalis 10C1 (Enterococcus faecalis) has been determined by crystal soaking, X-ray data collection, model building of NADH and refinement at 0.24-nm resolution based on the known enzyme structure [Stehle, T., Ahmed, S. A., Claiborne, A. & Schulz, G. E. (1991) J. Mol. Biol. 221, 1325-1344]. Apart from NADH, the catalytic center of the enzyme contains FAD and a cysteine that shuttles between thiolate and sulfenic acid states. Unfortunately, this cysteine was irreversibly oxidized to a cysteine sulfonic acid in the established enzyme structure. Based on the geometry of the catalytic center, we discuss the stabilization of the oxidation-sensitive sulfenic acid and propose a reaction mechanism.

Binding Sites↗

PubMed1993

Crystal structures of two mutants of adenylate kinase from Escherichia coli that modify the Gly-loop.

Two mutants of adenylate kinase from Escherichia coli have been crystallized and analyzed by X-ray diffraction at resolutions of 3.4 and 2.4 A, respectively. These mutants are Pro-9-->Leu and Gly-10-->Val. They were selected for their positions in the highly conserved Gly-loop forming a giant anion hole for the beta-phosphate of ATP (GTP) in adenylate kinases, H-ras-p21, and other nucleotide-binding proteins. Mutants at these positions of H-ras-p21 cause cancer. In adenylate kinase these mutations cause smallish changes at the active site. Relating the structural changes to the known changes in catalysis indicates that these mutants hinder the induced-fit movements. As a side result we find that mutant Pro-9-->Leu and wild-type form one very similar crystal packing contact that is crystallographic in one case and noncrystallographic in the other, while all other packing contacts and the space groups are quite at variance.

Adenylate Kinase↗

PubMed1993

Bacterial porins: structure and function.

Within the class of integral membrane proteins, the bacterial porins display a remarkable resistance to denaturants and proteases. This stability is probably crucial for the formation of highly ordered, three-dimensional crystals. Structural analysis of these crystals has been possible in atomic detail. This analysis has revealed interesting features, such as the aromatic girdles, and has helped to explain several observations, including the porins' ability to discriminate between polar and non-polar solutes. Recent research has thus improved our understanding of the porins in a qualitative fashion.

Animals↗

PubMed1992

Catalytic center of cyclodextrin glycosyltransferase derived from X-ray structure analysis combined with site-directed mutagenesis.

An X-ray structure analysis of a crystal of mutant Asp229----Ala of cyclodextrin glycosyltransferase from Bacillus circulans (Ec 2.4.1.19) that had been shortly exposed to beta-cyclodextrin showed density corresponding to a maltose bound at the catalytic center. The crystal structure was refined to an R-factor of 18.7% at 2.5-A resolution. The catalytic center is defined by homology with the structurally known alpha-amylases and by the observation that mutants Asp229----Ala and Asp328----Ala are almost inactive. By model building, the density-defined maltose was extended to a full beta-cyclodextrin, which then indicated the general locations of seven subsites for glucosyl units. The catalytically competent residues Asp229, Glu257, and Asp328 are at the reducing end of the density-defined maltose. In the unligated wild-type structure, Glu257 and Asp328 form a 2.6-A hydrogen bond between their carboxylates in an arrangement that resembles those of the catalytically competent carboxylates in acid proteases. Presumably, the first catalytic step is an attack of the proton between Glu257 and Asp328 on the oxygen of the glycosidic bond.

Bacillus↗