The solution structure of oxidized Escherichia coli cytochrome b562.

The solution structure of the oxidized, paramagnetic form of cytochrome b562 from Escherichia coli (106 amino acids) is here reported as obtained from 1653 meaningful NOEs (from a total of 2051 unique NOEs), 33 (3)JHNHalpha values, and 339 pseudocontact shifts. The structure displays the typical four-helix bundle motif, and a disordered loop between helices alpha2 and alpha3, as found in the solid state. The solution structure has a conformation intermediate between the two independent solid-state molecules, although different orientations are observed for a few residues. The magnetic susceptibility tensor is similar to that of cytochrome c, which has the same ligands, although the anisotropy is somewhat smaller. This difference in the electronic structure is consistent with the thermal accessibility in cytochrome b562 of states with S > 1/2. The structure is also compared with the solution structure of the apoprotein, and some information on the role of the cofactor on the protein folding and mobility is obtained. Helix alpha4 seems to be the most sensitive to the chemical environment in terms of structure and mobility. The pKa values affecting the hyperfine-shifted signals are also discussed. Quite intriguing is the comparison of the structure of cytochrome b562 with the available structures of cytochromes c' which display a similar folding motif and similar pKa values but very little sequence similarity.     

The molecular structure of UDP-galactose 4-epimerase from Escherichia coli determined at 2.5 A resolution.

UDP-galactose 4-epimerase catalyzes the conversion of UDP-galactose to UDP-glucose during normal galactose metabolism. The molecular structure of UDP-galactose 4-epimerase from Escherichia coli has now been solved to a nominal resolution of 2.5 A. As isolated from E. coli, the molecule is a dimer of chemically identical subunits with a total molecular weight of 79,000. Crystals of the enzyme used for this investigation were grown as a complex with the substrate analogue, UDP-benzene, and belonged to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 76.3 A, b = 83.1 A, c = 132.1 A, and one dimer per asymmetric unit. An interpretable electron density map calculated to 2.5 A resolution was obtained by a combination of multiple isomorphous replacement with six heavy atom derivatives, molecular averaging, and solvent flattening. Each subunit of epimerase is divided into two domains. The larger N-terminal domain, composed of amino acid residues 1-180, shows a classic NAD+ binding motif with seven strands of parallel beta-pleated sheet flanked on either side of alpha-helices. The seventh strand of the beta-pleated sheet is contributed by amino acid residues from the smaller domain. In addition, this smaller C-terminal domain, consisting of amino acid residues 181-338, contains three strands of beta-pleated sheet, two major alpha-helices and one helical turn. The substrate analogue, UDP-benzene, binds in the cleft located between the two domains with its phenyl ring in close proximity to the nicotinamide ring of NAD+. Contrary to the extensive biochemical literature suggesting that epimerase binds only one NAD+ per functional dimer, the map clearly shows electron density for two nicotinamide cofactors binding in symmetry-related positions in the dimer. Likewise, each subunit in the dimer also binds one substrate analogue.     

Crystallographic study of cytochrome b 562 from Escherichia coli

Three crystalline forms of cytochrome b₅₆₂ from Escherichia coli have been grown. One form, suitable for a three-dimensional structure investigation, is triclinic, containing two molecules per cell, with the following parameters: a = 33·71 Å, b = 50·51 Å, c = 32·71 Å, α = 102.62°, β = 86.67° and γ = 107.03°. X-ray data from the native crystals have been collected to 2.8 Å resolution and a search for suitable heavy atom derivatives has begun.     

Projection structure of the cytochrome bo ubiquinol oxidase from Escherichia coli at 6 A resolution.

The haem-copper cytochrome oxidases are terminal catalysts of the respiratory chains in aerobic organisms. These integral membrane protein complexes catalyse the reduction of molecular oxygen to water and utilize the free energy of this reaction to generate a transmembrane proton gradient. Quinol oxidase complexes such as the Escherichia coli cytochrome bo belong to this superfamily. To elucidate the similarities as well as differences between ubiquinol and cytochrome c oxidases, we have analysed two-dimensional crystals of cytochrome bo by cryo-electron microscopy. The crystals diffract beyond 5 A. A projection map was calculated to a resolution of 6 A. All four subunits can be identified and single alpha-helices are resolved within the density for the protein complex. The comparison with the three-dimensional structure of cytochrome c oxidase shows the clear structural similarity within the common functional core surrounding the metal-binding sites in subunit I. It also indicates subtle differences which are due to the distinct subunit composition. This study can be extended to a three-dimensional structure analysis of the quinol oxidase complex by electron image processing of tilted crystals.     

Crystal structure of Escherichia coli methionyl-tRNA synthetase at 2.5 Å resolution

Native methionyl-tRNA synthetase from Escherichia coli (a dimer of molecular weight 172,000) can be converted by mild proteolysis into a well-defined monomeric fragment of molecular weight 64,000. This fragment retains full specificity towards methionine and tRNA^Met, and has unimpaired activity in both the activation and aminoacylation reactions.This paper describes the structure of the active fragment, as determined by an X-ray crystallographic study at 2.5 Å resolution using five heavy-atom derivatives. The elongated molecule (90 Å × 52 Å × 44 Å) contains several α-helices, which account for 43% of the residues. Three domains can be distinguished in the structure: (1) a central core beginning at the N-terminus, consisting of a five-stranded parallel pleated sheet with α-helices connecting the β-strands; (2) a second domain with less-ordered structure, inserted between the third and fourth strand of the central sheet; (3) a C-terminal domain, beginning after the fifth parallel strand, very rich in α-helices.These three domains are organized in a biglobular structure; one globule contains the first and the second domain (N-terminal globule), the other the third domain. The two globules, linked together by a single chain, are separated by a large cleft.The most salient feature of the structure is the presence, in the N-terminal domain, of a “nucleotide binding fold” similar to that first observed in dehydrogenases. This makes methionyl-tRNA synthetase, and possibly all aminoacyl-tRNA synthetases, a new member of this family of nucleotide binding proteins possessing the characteristic “Rossmann fold”.     

The structure of cytochrome c3 from Desulfovibrio vulgaris Miyazaki at 2.5 A resolution

The structure of tetraheme cytochrome c3 isolated from Desulfovibrio vulgaris Miyazaki has been determined at 2.5 A resolution by an X-ray diffraction method. Protein phases were computed by the multiple isomorphous replacement method using the native and four heavy atom derivatives, anomalous scattering measurements of the latter being considered. The mean figure of merit was 0.77. Four heme groups are exposed on the surface of the molecule. There are some short helical segments in the polypeptide chain, and hair-pin turns are often observed at glycine and alanine residues.     

Crystal structure of Azotobacter cytochrome c5 at 2.5 A resolution

The crystal structure of cytochrome c5 from Azotobacter vinelandii has been solved and refined to an R value of 0.29 at 2.5 A resolution. The structure of the oxidized protein was solved using a monoclinic crystal form. The structure was solved by multiple isomorphous replacements, re-fit to a solvent-leveled multiple isomorphous replacement map, and refined by restrained least squares. The structure reveals monomers associated about the crystallographic 2-fold axis by hydrophobic contacts at the "exposed heme edge". The overall conformation for the monomer is similar to that of Pseudomonas aeruginosa cytochrome c551. However, relative to a common heme conformation, c5 and c551 differ by an average of 6.8 A over 82 alpha-carbon positions and the propionates of c5 are much more exposed to solvent. The shortest heme--heme contact at the "dimer" interface is 6.3 A (Fe to Fe 16.4 A). Alignment of c5 and c551 shows that the two cytochromes, in spite of sequence differences, have remarkably similar charge distributions. A disulfide stacks on a tyrosine between the N- and C-terminal helices.     

The three-dimensional structure of glutathione reductase from Escherichia coli at 3.0 A resolution

The structure of glutathione reductase from Escherichia coli has been solved at 3 A resolution using multiple isomorphous replacement, solvent flattening, and molecular replacement on the basis of the homologous (53% identical residues) and structurally well-established human enzyme. The structures of both enzyme species agree with each other in a global way; there is no domain rearrangement. In detail, clear structural differences can be observed. The structure analysis of the E. coli enzyme was tackled in order to understand site-directed mutants, the most spectacular of which changed the cofactor specificity of this enzyme from NADP to NAD (Scrutton et al., 1990, Nature 343:38-43).     

Crystal structure of thioredoxin from Escherichia coli at 1.68 A resolution

The crystal structure of thioredoxin from Escherichia coli has been refined by the stereochemically restrained least-squares procedure to a crystallographic R-factor of 0.165 at 1.68 A resolution. In the final model, the root-mean-square deviation from ideality for bond distances is 0.015 A and for angle distances 0.035 A. The structure contains 1644 protein atoms from two independent molecules, two Cu2+, 140 water molecules and seven methylpentanediol molecules. Ten residues have been modeled in two alternative conformations. E. coli thioredoxin is a compact molecule with 90% of its residues in helices, beta-strands or reverse turns. The molecule consists of two conformational domains, beta alpha beta alpha beta and beta beta alpha, connected by a single-turn alpha-helix and a 3(10) helix. The beta-sheet forms the core of the molecule packed on either side by clusters of hydrophobic residues. Helices form the external surface. The active site disulfide bridge between Cys32 and Cys35 is located at the amino terminus of the second alpha-helix. The positive electrostatic field due to the helical dipole is probably important for stabilizing the anionic intermediate during the disulfide reductase function of the protein. The more reactive cysteine, Cys32, has its sulfur atom exposed to solvent and also involved in a hydrogen bond with a backbone amide group. Residues 29 to 37, which include the active site cysteine residues, form a protrusion on the surface of the protein and make relatively fewer interactions with the rest of the structure. The disulfide bridge exhibits a right-handed conformation with a torsion angle of 81 degrees and 72 degrees about the S-S bond in the two molecules. Twenty-five pairs of water molecules obey the noncrystallographic symmetry. Most of them are involved in establishing intramolecular hydrogen-bonding interactions between protein atoms and thus serve as integral parts of the folded protein structure. Methylpentanediol molecules often pack against the loops and stabilize their structure. Cu2+ used for crystallization exhibit a distorted octahedral square bipyramid co-ordination and provide essential packing interactions in the crystal. The two independent protein molecules are very similar in conformation but distinctly different in atomic detail (root-mean-square = 0.94 A). The differences, which may be related to the crystal contacts, are localized mostly to regions far from the active site.     

Molecular structure of cytochrome c2 isolated from Rhodobacter capsulatus determined at 2.5 A resolution.

The molecular structure of the cytochrome c2, isolated from the purple photosynthetic bacterium Rhodobacter capsulatus, has been solved to a nominal resolution of 2.5 A and refined to a crystallographic R-factor of 16.8% for all observed X-ray data. Crystals used for this investigation belong to the space group R32 with two molecules in the asymmetric unit and unit cell dimensions of a = b = 100.03 A, c = 162.10 A as expressed in the hexagonal setting. An interpretable electron density map calculated at 2.5 A resolution was obtained by the combination of multiple isomorphous replacement with four heavy atom derivatives, molecular averaging and solvent flattening. At this stage of the structural analysis the electron densities corresponding to the side-chains are well ordered except for several surface lysine, glutamate and aspartate residues. Like other c-type cytochromes, the secondary structure of the protein consists of five alpha-helices forming a basket around the heme prosthetic group with one heme edge exposed to the solvent. The overall alpha-carbon trace of the molecule is very similar to that observed for the bacterial cytochrome c2, isolated from Rhodospirillum rubrum, with the exception of a loop, delineated by amino acid residues 21 to 32, that forms a two stranded beta-sheet-like motif in the Rb. capsulatus protein. As observed in the eukaryotic cytochrome c proteins, but not in the cytochrome c2 from Rsp. rubrum, there are two evolutionarily conserved solvent molecules buried within the heme binding pocket.     

Differences in thermal stability between reduced and oxidized cytochrome b562 from Escherichia coli

The thermal stabilities of ferri- and ferrocytochrome b562 were examined. Thermally induced spectral changes, monitored by absorption and second-derivative spectroscopies, followed the dissociation of the heme moiety and the increased solvation of tyrosine residue(s) located in close proximity to the heme binding site. All observed thermal transitions were independent of the rate of temperature increase (0.5-2 degrees C/min), and the denatured protein exhibited partial to near-complete reversibility upon return to ambient temperature. The extent of renaturation of cytochrome b562 is dependent on the amount of time the unfolded conformer is exposed to temperatures above the transition temperature, Tm. All thermally induced spectra changes fit a simple two-state model, and the thermal transition was assumed to be reversible. The thermal transition for ferrocytochrome b562 yielded Tm and van't Hoff enthalpy (delta HvH) values of 81.0 degrees C and 137 kcal/mol, respectively. In contrast, Tm and delta HvH values obtained for the ferricytochrome were 66.7 degrees C and 110 kcal/mol, respectively. The estimated increase in the stabilization free energy at the Tm of ferricytochrome b562 following the one-electron reduction to the ferrous form, where delta delta G = delta Tm delta Sm [delta Sm = 324 cal/(K.mol), delta Tm = 14.3 degrees C] [Becktel, W. J., & Schellman, J. A. (1987) Biopolymers 26, 1859-1877], is 4.6 kcal/mol.     

Refined structure of alkaline phosphatase from Escherichia coli at 2.8 A resolution

The structure of alkaline phosphatase from Escherichia coli has been determined to 2.8 A resolution. The multiple isomorphous replacement electron density map of the dimer at 3.4 A was substantially improved by molecular symmetry averaging and solvent flattening. From these maps, polypeptide chains of the dimer were built using the published amino acid sequence. Stereochemically restrained least-squares refinement of this model against native data, starting with 3.4 A data and extending in steps to 2.8 A resolution, proceeded to a final overall crystallographic R factor of 0.256. Alkaline phosphatase-phosphomonoester hydrolase (EC 3.1.3.1) is a metalloenzyme that forms an isologous dimer with two reactive centers 32 A apart. The topology of the polypeptide fold of the subunit is of the alpha/beta class of proteins. Despite the similarities in the overall alpha/beta fold with other proteins, alkaline phosphatase does not have a characteristic binding cleft formed at the carboxyl end of the parallel sheet, but rather an active pocket that contains a cluster of three functional metal sites located off the plane of the central ten-stranded sheet. This active pocket is located near the carboxyl ends of four strands and the amino end of the antiparallel strand, between the plane of the sheet and two helices on the same side. Alkaline phosphatase is a non-specific phosphomonoesterase that hydrolyzes small phosphomonoesters as well as the phosphate termini of DNA. The accessibility calculations based on the refined co-ordinates of the enzyme show that the active pocket barely accommodates inorganic phosphate. Thus, the alcoholic or phenolic portion of the substrate would have to be exposed on the surface of the enzyme. Two metal sites, M1 and M2, 3.9 A apart, are occupied by zinc. The third site, M3, 5 A from site M2 and 7 A from site M1, is occupied by magnesium or, in the absence of magnesium, by zinc. As with other zinc-containing enzymes, histidine residues are ligands to zinc site M1 (three) and to zinc site M2 (one). Ligand assignment and metal preference indicate that the crystallographically found metal sites M1, M2 and M3 correspond to the spectroscopically deduced metal sites A, B and C, respectively. Arsenate, a product analog and enzyme inhibitor, binds between Ser102 and zinc sites M1 and M2. The position of the guanidinium group of Arg 166 is within hydrogen-bonding distance from the arsenate site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Three-dimensional structure of aspartate aminotransferase from Escherichia coli at 2.8 A resolution

The crystal structure of aspartate aminotransferase of Escherichia coli was determined by X-ray structure analysis at 2.8 A resolution. The structure was solved by the molecular replacement method and refined to an R-factor of 0.27, and it was found that the overall structure of AspAT of E. coli is similar to that of those of higher animals.     

Three-dimensional structure of thymidine phosphorylase from Escherichia coli at 2.8 A resolution

The three-dimensional structure of thymidine phosphorylase from Escherichia coli has been determined at 2.8 A resolution using multiple-isomorphous-replacement techniques. The amino acid sequence deduced from the deoA DNA sequence is also reported. Thymidine phosphorylase exists in the crystal as an S-shaped dimer in which the subunits are related by a crystallographic 2-fold axis. Each subunit is composed of a small alpha-helical domain of six helices and a large alpha/beta domain. The alpha/beta domain includes a six-stranded mixed beta-sheet and a four-stranded antiparallel beta-sheet. The active site has been identified by difference Fourier analyses of the binding of thymine and thymidine and lies in a cavity between the small and large domains. The central beta-sheet is splayed open to accommodate a putative phosphate-binding site which is probably occupied by a sulfate ion in the crystal.     

Crystal structure of Escherichia coli cystathionine gamma-synthase at 1.5 A resolution.

The transsulfuration enzyme cystathionine gamma-synthase (CGS) catalyses the pyridoxal 5'-phosphate (PLP)-dependent gamma-replacement of O-succinyl-L-homoserine and L-cysteine, yielding L-cystathionine. The crystal structure of the Escherichia coli enzyme has been solved by molecular replacement with the known structure of cystathionine beta-lyase (CBL), and refined at 1.5 A resolution to a crystallographic R-factor of 20.0%. The enzyme crystallizes as an alpha4 tetramer with the subunits related by non-crystallographic 222 symmetry. The spatial fold of the subunits, with three functionally distinct domains and their quaternary arrangement, is similar to that of CBL. Previously proposed reaction mechanisms for CGS can be checked against the structural model, allowing interpretation of the catalytic and substrate-binding functions of individual active site residues. Enzyme-substrate models pinpoint specific residues responsible for the substrate specificity, in agreement with structural comparisons with CBL. Both steric and electrostatic designs of the active site seem to achieve proper substrate selection and productive orientation. Amino acid sequence and structural alignments of CGS and CBL suggest that differences in the substrate-binding characteristics are responsible for the different reaction chemistries. Because CGS catalyses the only known PLP-dependent replacement reaction at Cgamma of certain amino acids, the results will help in our understanding of the chemical versatility of PLP.     

X-ray crystal structure of aminoimidazole ribonucleotide synthetase (PurM), from the Escherichia coli purine biosynthetic pathway at 2.5 A resolution.

BACKGROUND: The purine biosynthetic pathway in procaryotes enlists eleven enzymes, six of which use ATP. Enzymes 5 and 6 of this pathway, formylglycinamide ribonucleotide (FGAR) amidotransferase (PurL) and aminoimidazole ribonucleotide (AIR) synthetase (PurM) utilize ATP to activate the oxygen of an amide within their substrate toward nucleophilic attack by a nitrogen. AIR synthetase uses the product of PurL, formylglycinamidine ribonucleotide (FGAM) and ATP to make AIR, ADP and P(i). RESULTS: The structure of a hexahistidine-tagged PurM has been solved by multiwavelength anomalous diffraction phasing techniques using protein containing 28 selenomethionines per asymmetric unit. The final model of PurM consists of two crystallographically independent dimers and four sulfates. The overall R factor at 2.5 A resolution is 19.2%, with an R(free) of 26.4%. The active site, identified in part by conserved residues, is proposed to be a long groove generated by the interaction of two monomers. A search of the sequence databases suggests that the ATP-binding sites between PurM and PurL may be structurally conserved. CONCLUSIONS: The first structure of a new class of ATP-binding enzyme, PurM, has been solved and a model for the active site has been proposed. The structure is unprecedented, with an extensive and unusual sheet-mediated intersubunit interaction defining the active-site grooves. Sequence searches suggest that two successive enzymes in the purine biosynthetic pathway, proposed to use similar chemistries, will have similar ATP-binding domains.     

Improvement of the 2.5 Å resolution model of cytochrome b562 by redetermining the primary structure and using molecular graphics

The amino acid sequence of cytochrome b₅₆₂ has been redetermined and fitted to a 2.5 Å electron density map. A combination of cyanogen bromide fragmentation, proteolytic cleavages and manual and automatic sequencing was used. The model was built on an MMS-X molecular graphics system by interactively fitting the model to the electron density.The results indicate that the protein is 106 rather than 110 residues in length. The largest change required has been the rearrangement and modification of a continuous stretch of 11 residues of the original sequence. The electron density map confirms most of these changes, some of which were proposed before the revised sequence became available.