Biomedical subjects

Z Dauter

Publications and source records attributed to Z Dauter.

At least 91 records · Page 5Linked to original sources

PubMed1995

Refined structure of Cu-substituted alcohol dehydrogenase at 2.1 A resolution.

Liver alcohol dehydrogenase (LADH) is a Zn(II)-dependent dimeric enzyme. LADH with the active-site Zn(II) substituted by Cu(II) resembles blue (type I) copper proteins by its spectroscopic characteristics. In this work we present the X-ray structure of the active site Cu(II)-substituted LADH complex with NADH and dimethyl sulfoxide (DMSO). The structure was solved by molecular replacement. The space group is P2(1) with cell dimensions a = 44.4, b = 180.6, c = 50.8 A and beta = 108 degrees. There is one dimer of the enzyme in the asymmetric unit. The refinement was carried out to a crystallographic R-factor of 16.1% for 41 119 unique reflections in the resolution range 12.0 to 2.1 A. The coordination geometry of Cu(II) in LADH is compared with the active-site metal coordination in the Zn-LADH-NADH-DMSO complex and blue-copper proteins. The distances from the metal to the protein ligands (Cys46, His67 and Cys174) are similar for the Zn(II) and Cu(II) ions. The distances of the O atom of the inhibitor DMSO to the Cu(II) ion in the two subunits of the dimer are 3.19 and 3.45 A. These are considerably longer than the corresponding distances for the Zn(II) enzyme, 2.19 and 2.15 A. The Cu(II) ion is positioned nearly in the plane of the three protein ligands (NS(2)) with a geometry similar to the trigonal arrangement of the three strongly bound ligands (N(2)S) in blue-copper proteins. This coordination probably accounts for the similarity of the spectral characteristics of Cu(II)-LADH and type I copper proteins.

Journal Article↗

PubMed1995

The crystal structure of apo-pseudoazurin from Alcaligenes faecalis S-6.

The 3D structure of the apo-pseudoazurin (copper free pseudoazurin) from Alcaligenes faecalis strain S-6 is determined and refined at pH 6.7 using X-ray diffraction data to 1.85 A resolution. The final crystallographic R-factor is 0.164. Comparing the structures of apo-pseudoazurin and the native (Cu2+) protein, we observed limited differences ranging between 0.1-0.4 A at the vicinity of the copper site, at the loops connecting the secondary structural elements, at certain beta-strands and at the amino and carboxy termini of the protein.

Alcaligenes↗

PubMed1995

The Escherichia coli malonyl-CoA:acyl carrier protein transacylase at 1.5-A resolution. Crystal structure of a fatty acid synthase component.

Endogenous fatty acids are synthesized in all organisms in a pathway catalyzed by the fatty acid synthase complex. In bacteria, where the fatty acids are used primarily for incorporation into components of cell membranes, fatty acid synthase is made up of several independent cytoplasmic enzymes, each catalyzing one specific reaction. The initiation of the elongation step, which extends the length of the growing acyl chain by two carbons, requires the transfer of the malonyl moiety from malonyl-CoA onto the acyl carrier protein. We report here the crystal structure (refined at 1.5-A resolution to an R factor of 0.19) of the malonyl-CoA specific transferase from Escherichia coli. The protein has an alpha/beta type architecture, but its fold is unique. The active site inferred from the location of the catalytic Ser-92 contains a typical nucleophilic elbow as observed in alpha/beta hydrolases. Serine 92 is hydrogen bonded to His-201 in a fashion similar to various serine hydrolases. However, instead of a carboxyl acid typically found in catalytic triads, the main chain carbonyl of Gln-250 serves as a hydrogen bond acceptor in an interaction with His-201. Two other residues, Arg-117 and Glu-11, are also located in the active site, although their function is not clear.

Acyl-Carrier Protein S-Malonyltransferase↗

PubMed1995

Crystal structure of the closed form of chicken cytosolic aspartate aminotransferase at 1.9 A resolution.

The crystal structure of chicken cytosolic aspartate aminotransferase (cAATase; EC 2.6.1.1) has been solved and refined at 1.9 A resolution. Orthorhombic crystals, space group P2(1)2(1)2(1), a = 56.4 A, b = 126.0 A and c = 142.3 A, were grown from polyethylene glycol solutions in the presence of maleate, a dicarboxylic inhibitor that forms a Michaelis-like complex. The pyridoxal form of the enzyme was used for crystallization. Diffraction data were collected using synchrotron radiation. The structure of the new orthorhombic crystal form was solved by molecular replacement using the partially refined 2.8 A resolution structure of the high-salt crystal form as a search model. The final value of the crystallographic R-factor after rigid body and restrained least-squares refinement is 0.175 with very good model geometry. The two 2-fold-related subunits of cAATase have distinct environments in the crystal lattice. Domain movement is strictly hindered by the lattice contacts in one subunit, while the second one possesses conformational freedom. Despite their different environments, both subunits were found in the closed conformation with one maleate molecule tightly bound in each active site. The present study allows a detailed comparison of the highly refined structures of the aspartate aminotransferase isozymes, and thus provide better insight into the role of conserved and variable residues in substrate recognition and catalysis.

Animals↗

PubMed1995

Crystal structure of narbonin at 1.8 A resolution.

The three-dimensional structure of narbonin, a seed protein from Vicia narbonensis L, has been determined at 1.8 A resolution. Phase information was obtained by multiple isomorphous replacement and optimized anomalous dispersion. The narbonin structure was initially traced with only 17% amino-acid sequence information and preliminarily refined to a crystallographic R-factor of 16.5%. It is now refined to 15.9% using full sequence information derived from cDNA and after the addition of more solvent molecules. The monomeric molecule of narbonin is an eight-stranded parallel beta-barrel surrounded by alpha-helices in a beta/alpha-topology similar to that first observed in triose phosphate isomerase. Differences exist in the N-terminal part of the polypeptide chain, where the first helix is replaced by a loop and the second beta-strand is followed by an additional antiparallel alpha-sheet placed parallel on top of alpha-helices alpha3 and alpha4. Two short additional secondary structures are present. The first, an alpha-helix, is situated between the seventh beta-strand and the following helix, and the second, which is a 3(10) helix, between the eighth strand and the C-terminal helix. The most striking observation is the lack of a known enzymatic function for narbonin, because all TIM-like structures known so far are enzymes.

Journal Article↗

PubMed1995

Purification, crystallisation and preliminary X-ray analysis of the vanadium-dependent haloperoxidase from Corallina officinalis.

The vanadium-dependent haloperoxidase from the seaweed Corallina officinalis has been purified to homogeneity and crystallised. The protein is reported to be a hexamer of 12 x 64,000 Da, contains no haem, and is dependent on vanadium for activity. The crystals are grown from polyethylene glycol (PEG) 6,000 and 0.4 M potassium chloride. They are stable and diffract to better than 2 A resolution. They are of a cubic space group I23 (or 12(1)3) with cell dimensions a = b = c = 310 A.

Crystallization↗

PubMed1995

Structure of octreotide, a somatostatin analogue.

Octreotide, a synthetic somatostatin analogue, is an octapeptide with one disulfide bridge. Crystals of octreotide are orthorhombic, space group P2(1)2(1)2(1), a = 18.458 (5), b = 30.009 (7), c = 39.705 (27) A, with three molecules of octapeptide, one ordered oxalate dianion and 52 water molecules in the asymmetric unit. Complete protonation of the NH(2) groups (as assumed in the refinement) would require three oxalate dianions in the asymmetric unit for charge neutrality; a chemical analysis indicated that four are present. In either case they are so disordered that they cannot be distinguished from the water molecules. The 18 951 unique reflections (R(sym) = 0.026) used for structure solution and refinement were recorded with the EMBL imaging-plate scanner using synchrotron radiation. The structure was solved by Patterson interpretation, locating the three disulfide bridges, followed by tangent phase expansion and E-Fourier recycling. The anisotropic refinement against all F(2) data between 1.04 and 10.0 A resolution by blocked restrained full-matrix least-squares techniques converged to a conventional R index based on F of 0.084 [I > 2a(I) and 10.0 > d > 1.04 A] and wR2, the weighted R-index on F(2), of 0.246 (for all data). One peptide molecule adopts a flat beta-sheet structure; the other two possess different irregular backbone conformations, but are similar to each other. All three molecules have a distorted type II' beta-turn around the D-Trp-Lys region, but exhibit different side-chain conformations. The crystal structure is stabilized by a network of inter- and intramolecular hydrogen bonds.

Journal Article↗

PubMed1995

Comparison of different X-ray data-collection systems using the crystal structure of octreotide.

The octapeptide octreotide crystallizes with three peptide molecules and about 20% water in the asymmetric unit, and in many ways possesses diffraction properties similar to those of a 'mini-protein' consisting of 24 amino-acid residues. It diffracts to about 1.0 A but data in the range 1.4-1.0 A are weak. It provides a suitable test of different macromolecular X-ray data-collection techniques, especially of their ability to measure weak reflections accurately. In contrast to typical proteins it is possible to perform a full anisotropic refinement, that we believe provides a more objective test of the quality of the data than the internal consistency of equivalent reflections. We have collected a total of six data sets. The X-ray sources included synchrotron radiation, Cu Kalpha rotating anodes and Mo Kalpha sealed tubes; position-sensitive two-dimensional detectors from four manufacturers and a four-circle diffractometer with scintillation counter were employed. Two of the six data sets were collected at low temperature. Reasonable anisotropic refinement was possible with all area-detector data sets, although significant differences in the precision of the final model were observed. In addition we tested the ability of automated Patterson interpretation to solve the structure using the six independent data sets. The structure solution was only successful using the synchrotron or rotating-anode data sets, i.e. for the more intense sources. It appears that for structure solution the maximum resolution of the data is critical, whereas for refinement the accuracy of the data is more important.

Journal Article↗

PubMed1995

Proteins at atomic resolution.

Experimental advances in data collection, including bright sources, cryogenic cooling and two-dimensional detectors, have made it tractable to record data to beyond 1.2 A for several proteins, yielding high-accuracy models and fine details of structure. For small metalloproteins, atomic-resolution data have enabled ab initio solution of the phase problem.

Computers↗

PubMed1995

How nature deals with stereoisomers.

All natural proteins are composed of L-amino acids and are inherently chiral. The properties of both L- and chemically synthesized D-amino acids are identical except in optically asymmetric interactions. Structural studies of D-I racemic mixtures of crystallographic interest are discussed. The review also gives some recent examples of stereospecificity: how L-proteins deal with L- or D-substrates and how enzymes can function as racemases. Two particular examples of stereoselectivity are then discussed.

Amino Acids↗

PubMed1995

A common protein fold and similar active site in two distinct families of beta-glycanases.

The structure of Clostridium thermocellum endoglucanase CelC, a member of the largest cellulase family (family A), has been determined at 2.15 A resolution. The protein folds into an (alpha/beta)8 barrel, with a deep active-site cleft generated by the insertion of a helical subdomain. The structure of the catalytic core of xylanase XynZ, which belongs to xylanase family F, has been determined at 1.4 A resolution. In spite of significant differences in substrate specificity and structure (including the absence of the helical subdomain), the general polypeptide folding pattern, architecture of the active site and catalytic mechanism of XynZ and CelC are similar, suggesting a common evolutionary origin.

Amino Acid Sequence↗

PubMed1994

Crystal structure of a bacterial chitinase at 2.3 A resolution.

BACKGROUND: Chitinases cleave the beta-1-4-glycosidic bond between the N-acetyl-D-glucosamine units of which chitin is comprised. Chitinases are present in plants, bacteria and fungi, but whereas structures are available for two prototypic plant enzymes, no structure is available for a bacterial or fungal chitinase. RESULTS: To redress this imbalance, the structure of native chitinase A from Serratia marcescens has been solved by multiple isomorphous replacement and refined at 2.3 A resolution, resulting in a crystallographic R-factor of 16.2%. The enzyme comprises three domains: an all beta-strand amino-terminal domain, a catalytic alpha/beta-barrel domain, and a small alpha+beta-fold domain. There are several residues with unusual geometries in the structure. Structure determination of chitinase A in complex with N,N',N",N"'-tetra-acetylo-chitotetraose, together with biochemical and sequence analysis data, enabled the positions of the active-site and catalytic residues to be proposed. CONCLUSIONS: The reaction mechanism seems to be similar to that of lysozyme and most other glycosylhydrolases, i.e. general acid-base catalysis. The role of the amino-terminal domain could not be identified, but it has similarities to the fibronectin III domain. This domain may possibly facilitate the interaction of chitinase A with chitin.

Amino Acid Sequence↗

PubMed1994

11-fold symmetry of the trp RNA-binding attenuation protein (TRAP) from Bacillus subtilis determined by X-ray analysis.

The trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis has been crystallized and examined by crystallography using X-ray synchrotron radiation diffraction data. Crystals of TRAP complexed with L-tryptophan belong to space group C2 with a = 156.8 A, b = 114.05 A, c = 105.9 A, beta = 118.2 degrees. Crystals of a potential heavy-atom derivative of TRAP complexed with 5-bromo-L-tryptophan grow in the same space group with similar cell dimensions. X-ray data for the native crystals and for the derivative have been collected to 2.9 A and 2.2 A resolution, respectively. Peaks in the self-rotation function and in the Patterson synthesis could only be explained by two 11-subunit oligomers (each formed by an 11-fold axis of symmetry) in the asymmetric unit lying with the 11-fold rotation axes parallel to each other. The consequence is that the TRAP molecule has 11-fold symmetry and contains 11 subunits.

Bacillus subtilis↗

PubMed1994

2.2 A resolution structure of the amino-terminal half of HIV-1 reverse transcriptase (fingers and palm subdomains).

BACKGROUND: HIV-1 reverse transcriptase (RT) catalyzes the transformation of single-stranded viral RNA into double-stranded DNA, which is integrated into host cell chromosomes. The molecule is a heterodimer of two subunits, p51 and p66. The amino acid sequence of p51 is identical to the sequence of the amino-terminal subdomains of p66. Earlier crystallographic studies indicate that the RT molecule is flexible, which may explain the difficulty in obtaining high-resolution data for the intact protein. We have therefore determined the structure of a fragment of RT (RT216), which contains only the amino-terminal half of the RT molecule ('finger' and 'palm' subdomains). RESULTS: The crystal structure of RT216 has been refined at 2.2 A resolution to a crystallographic R-value of 20.8%. The structure is very similar to that of the corresponding part of the p66 subunit in the p66/p51 heterodimer, although there is a small difference in the relative orientation of the two subdomains compared with the structure of an RT-DNA-antibody fragment complex. There are a large number of stabilizing contacts (mainly hydrogen bonds and hydrophobic interactions) between the subdomains. The locations of conserved amino acids and the position of some important drug-resistant mutations are described. CONCLUSIONS: The RT216 structure provides detailed three-dimensional information of one important part of HIV-1 RT (including the critical active site residues). We propose a model to explain the inhibitory effect of non-nucleoside inhibitors, which partially accounts for their effect in terms of conformational changes of active site residues.

Binding Sites↗

PubMed1994

High-resolution structures of single-metal-substituted concanavalin A: the Co,Ca-protein at 1.6 A and the Ni,Ca-protein at 2.0 A.

The molecular structures of cobalt- and nickel-substituted concanavalin A have been refined at 1.6 and 2.0 A resolution, respectively. Both metal derivatives crystallize in space group I222 with approximate cell dimensions a = 89, b = 87 and c = 63 A and one monomer in the asymmetric unit. The final R factor for Co-substituted concanavalin A is 17.8% for 29 211 reflections with F > 1.0sigma(F) between 8.0 and 1.6 A. For Ni-substituted concanavalin A the final R factor is 15.9% for 16 128 reflections with F > 1.0sigma(F) between 8.0 and 2.0 A resolution. Both structures contain a transition-metal binding site and a calcium-binding site but, unlike Cd-substituted concanavalin A, do not have a third metal-binding site. The Co-substituted concanavalin A structure diffracts to the highest resolution of any concanavalin A structure reported to date. A comparison of the structures of Ni-, Co-, Cd-substituted and native concanavalin A gives an indication of coordinate errors, which is a useful baseline for comparisons with saccharide complexes of concanavalin A described in other work. We also give a detailed account of multiple conformations which were found for five side-chain residues.

Journal Article↗

PubMed1994

Crystallization and structure determination of bovine profilin at 2.0 A resolution.

Profilin regulates the behavior of the eukaryotic microfilament system through its interaction with non-filamentous actin. It also binds several ligands, including poly(L-proline) and the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Bovine profilin crystals (space group C2; a = 69.15 A, b = 34.59 A, c = 52.49 A; alpha = gamma = 90 degrees, beta = 92.56 degrees) were grown from a mixture of poly(ethylene glycol) 400 and ammonium sulfate. X-ray diffraction data were collected on an imaging plate scanner at the DORIS storage ring (DESY, Hamburg), and were phased by molecular replacement, using a search model derived from the 2.55 A structure of profilin complexed to beta-actin. The refined model of bovine profilin has a crystallographic R-factor of 16.5% in the resolution range 6.0 to 2.0 A and includes 128 water molecules, several of which form hydrogen bonds to stabilize unconventional turns. The structure of free bovine profilin is similar to that of bovine profilin complexed to beta-actin, and C alpha atoms from the two structures superimpose with an r.m.s. deviation of 1.25 A. This value is reduced to 0.51 A by omitting Ala1 and the N-terminal acetyl group, which lie at a profilin-actin interface in crystals of the complex. These residues display a strained conformation in crystalline profilin-actin but may allow the formation of a hydrogen bond between the N-acetyl carbonyl group of profilin and the phenol hydroxyl group of Tyr188 in actin. Several other actin-binding residues of profilin show different side-chain rotomer conformations in the two structures. The polypeptide fold of bovine profilin is generally similar to those observed by NMR for profilin from other sources, although the N terminus of Acanthamoeba profilin isoform I lies in a distorted helix and the C-terminal helix is less tilted with respect to the strands in the central beta-pleated sheet than is observed in bovine profilin. The majority of the aromatic residues in profilin are exposed to solvent and lie in either of two hydrophobic patches, neither of which takes part in an interface with actin. One of these patches is required for binding poly(L-proline) and contains an aromatic cluster comprising the highly conserved residues Trp3, Tyr6, Trp31 and Tyr139. In forming this cluster, Trp31 adopts a sterically strained rotamer conformation.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗

PubMed1994

Structure of the ADP complex of the 3-phosphoglycerate kinase from Bacillus stearothermophilus at 1.65 A.

The structure of the ADP complex of the enzyme 3-phosphoglycerate kinase (PGK, E.C. 2.7.2.3) from Bacillus stearothermophilus NCA-1503 has been determined by the method of molecular replacement. The structure has been refined to an R factor of 0.16 for all data between 10.0 and 1.65 A resolution, using data collected on the Hendrix-Lentfer imaging plate at the EMBL outstation in Hamburg. The r.m.s. deviations from stereochemical ideality are 0.010 and 0.011 A for bonds and planes, respectively. Although crystallized in the presence of the nucleotide product MgATP, the high-resolution structure reveals the bound nucleotide to be MgADP reflecting the low intrinsic ATPase activity of PGK. Although the two domains of this enzyme are found to be some 4.5 degrees closer together than is found in the yeast and horse-muscle apo-enzyme structures, this structure represents the 'open' rather than the 'closed', catalytically competent form, of the enzyme.

Journal Article↗

PubMed1994

High resolution structures of holo and apo formate dehydrogenase.

Three-dimensional crystal structures of holo (ternary complex enzyme-NAD-azide) and apo NAD-dependent dimeric formate dehydrogenase (FDH) from the methylotrophic bacterium Pseudomonas sp. 101 have been refined to R factors of 11.7% and 14.8% at 2.05 and 1.80 A resolution, respectively. The estimated root-mean-square error in atomic co-ordinates is 0.11 A for holo and 0.18 A for apo. X-ray data were collected from single crystals using an imaging plate scanner and synchrotron radiation. In both crystal forms there is a dimer in the asymmetric unit. Both structures show essentially 2-fold molecular symmetry. NAD binding causes movement of the catalytic domain and ordering of the C terminus, where a new helix appears. This completes formation of the enzyme active centre in holo FDH. NAD is bound in the cleft separating the domains and mainly interacts with residues from the co-enzyme binding domain. In apo FDH these residues are held in essentially the same conformation by water molecules occupying the NAD binding region. An azide molecule is located near the point of catalysis, the C4 atom of the nicotinamide moiety of NAD, and overlaps with the proposed formate binding site. There is an extensive channel running from the active site to the protein surface and this is supposed to be used by substrate to reach the active centre after NAD has already bound. The structure of the active site and a hypothetical catalytic mechanism are discussed. Sequence homology of FDH with other NAD-dependent formate dehydrogenases and some D-specific dehydrogenases is discussed on the basis of the FDH three-dimensional structure.

Amino Acid Sequence↗