Crystallization and preliminary X-ray characterization of a glycerol dehydrogenase from the human pathogen Salmonella enterica serovar Typhimurium.

Glycerol dehydrogenase (GldA) encoded by the STM4108 gene (gldA) has been related to the synthesis of HilA, a major transcriptional regulator that is responsible for the expression of invasion genes in the human pathogen Salmonella enterica serovar Typhimurium. Single colourless crystals were obtained from a recombinant preparation of GldA overexpressed in Escherichia coli. They belonged to space group P222(1), with unit-cell parameters a = 127.0, b = 160.1, c = 665.2 A. The crystals contained a very large number of molecules in the asymmetric unit, probably 30-35. Diffraction data were collected to 3.5 A resolution using synchrotron radiation at the European Synchrotron Radiation Facility.

Expression, purification, crystallization and preliminary X-ray characterization of two crystal forms of stationary-phase survival E protein from Campylobacter jejuni.

Survival E (SurE) protein from Campylobacter jejuni, a Gram-negative mesophile, has been overexpressed in Escherichia coli as a soluble protein, successfully purified and crystallized in two distinct crystal forms. The first form belongs to space group P2(1)2(1)2(1), with a tetramer in the asymmetric unit and unit-cell parameters a = 80.5, b = 119.0, c = 135.3 A. The second form belongs to space group C2, with unit-cell parameters a = 121.4, b = 47.1, c = 97.8 A, and contains a dimer in the asymmetric unit. Diffraction data have been collected from these crystal forms to 2.5 and 2.95 A resolution, respectively.

Crystallization and preliminary X-ray characterization of 1,3-propanediol dehydrogenase from the human pathogen Klebsiella pneumoniae.

1,3-Propanediol dehydrogenase (1,3-PD-DH), encoded by the dhaT gene, is a key enzyme in the dissimilation process for converting glycerol to 1,3-propanediol in the human pathogen Klebsiella pneumoniae. Single colourless crystals were obtained from a recombinant preparation of 1,3-propanediol dehydrogenase overexpressed in Escherichia coli. The crystals belong to space group P2(1), with unit-cell parameters a = 91.9, b = 226.6, c = 232.6 A, beta = 92.9 degrees. The crystals probably contain two decamers in the asymmetric unit, with a V(M) value of 3.07 A3 Da(-1) and an estimated solvent content of 59%. Diffraction data were collected to 2.7 A resolution using synchrotron radiation at the ID14-4 beamline of the European Synchrotron Radiation Facility.

Cloning, expression, purification, crystallization and preliminary structure determination of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate.

The cloning, expression, purification, crystallization and preliminary crystallographic analysis of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate are reported. Diffraction data sets were obtained from seven crystal forms in five different space groups, with highest resolutions ranging from 4.20 to 2.65 A. The phase problem was solved for a P2(1) crystal form using multiple isomorphous replacement with anomalous scattering from an osmium derivative and a SeMet derivative. The best native crystal in space group P2(1) has unit-cell parameters a = 105.5, b = 85.7, c = 151.8 A, beta = 105.2 degrees . Model building and refinement are currently under way.

The primary and three-dimensional structures of a nine-haem cytochrome c from Desulfovibrio desulfuricans ATCC 27774 reveal a new member of the Hmc family.

BACKGROUND: Haem-containing proteins are directly involved in electron transfer as well as in enzymatic functions. The nine-haem cytochrome c (9Hcc), previously described as having 12 haem groups, was isolated from cells of Desulfovibrio desulfuricans ATCC 27774, grown under both nitrate- and sulphate-respiring conditions. RESULTS: Models for the primary and three-dimensional structures of this cytochrome, containing 292 amino acid residues and nine haem groups, were derived using the multiple wavelength anomalous dispersion phasing method and refined using 1.8 A diffraction data to an R value of 17.0%. The nine haem groups are arranged into two tetrahaem clusters, with Fe-Fe distances and local protein fold similar to tetrahaem cytochromes c3, while the extra haem is located asymmetrically between the two clusters. CONCLUSIONS: This is the first known three-dimensional structure in which multiple copies of a tetrahaem cytochrome c3-like fold are present in the same polypeptide chain. Sequence homology was found between this cytochrome and the C-terminal region (residues 229-514) of the high molecular weight cytochrome c from Desulfovibrio vulgaris Hildenborough (DvH Hmc). A new haem arrangement in domains III and IV of DvH Hmc is proposed. Kinetic experiments showed that 9Hcc can be reduced by the [NiFe] hydrogenase from D. desulfuricans ATCC 27774, but that this reduction is faster in the presence of tetrahaem cytochrome c3. As Hmc has never been found in D. desulfuricans ATCC 27774, we propose that 9Hcc replaces it in this organism and is therefore probably involved in electron transfer across the membrane.

Ab initio determination of the crystal structure of cytochrome c6 and comparison with plastocyanin.

BACKGROUND: Electron transfer between cytochrome f and photosystem I (PSI) can be accomplished by the heme-containing protein cytochrome c6 or by the copper-containing protein plastocyanin. Higher plants use plastocyanin as the only electron donor to PSI, whereas most green algae and cyanobacteria can use either, with similar kinetics, depending on the copper concentration in the culture medium. RESULTS: We report here the determination of the structure of cytochrome c6 from the green alga Monoraphidium braunii. Synchrotron X-ray data with an effective resolution of 1.2 A and the presence of one iron and three sulfur atoms enabled, possibly for the first time, the determination of an unknown protein structure by ab initio methods. Anisotropic refinement was accompanied by a decrease in the 'free' R value of over 7% the anisotropic motion is concentrated at the termini and between residues 38 and 53. The heme geometry is in very good agreement with a new set of heme distances derived from the structures of small molecules. This is probably the most precise structure of a heme protein to date. CONCLUSIONS: On the basis of this cytochrome c6 structure, we have calculated potential electron transfer pathways and made comparisons with similar analyses for plastocyanin. Electron transfer between the copper redox center of plastocyanin to PSI and from cytochrome f is believed to involve two sites on the protein. In contrast, cytochrome c6 may well use just one electron transfer site, close to the heme unit, in its corresponding reactions with the same two redox partners.

X-ray structure of [Ru3 O2 (NH3)14]6+, cation of the cytological reagent Ruthenium Red.

The X-ray crystal structure of [Ru3 O2 (NH3)14] (S2 O3)3 . 4H2 O, the thiosulphate salt of Ruthenium Red, has been determined. The cation contains an essentially linear N-Ru-O-Ru-O-Ru-N backbone formed from three ruthenium coordination octahedra, giving an effectively cylindrical shape to the ion. Resonance Raman spectra are consistent with retention of this structure in solution.

The caa3 terminal oxidase of the thermohalophilic bacterium Rhodothermus marinus: a HiPIP:oxygen oxidoreductase lacking the key glutamate of the D-channel.

The respiratory chain of the thermohalophilic bacterium Rhodothermus marinus contains a novel complex III and a high potential iron-sulfur protein (HiPIP) as the main electron shuttle (Pereira et al., Biochemistry 38 (1999) 1268-1275 and 1276-1283). In this paper, one of the terminal oxidases expressed in this bacterium is extensively characterised. It is a caa3-type oxidase, isolated with four subunits (apparent molecular masses of 42, 19 and 15 kDa and a C-haem containing subunit of 35 kDa), which has haems of the A(s) type. This oxidase is capable of using TMPD and horse heart cytochrome c as substrates, but has a higher turnover with HiPIP, being the first example of a HiPIP:oxygen oxidoreductase. The oxidase has unusually low reduction potentials of 260 (haem C), 255 (haem A) and 180 mV (haem A3). Subunit I of R. marinus caa3 oxidase has an overall significant homology with the subunits I of the COX type oxidases, namely the metal binding sites and most residues considered to be functionally important for proton uptake and pumping (K- and D-channels). However, a major difference is present: the putative essential glutamate (E278 in Paraccocus denitrificans) of the D-channel is missing in the R. marinus oxidase. Homology modelling of the R. marinus oxidase shows that the phenol group of a tyrosine residue may occupy a similar spatial position as the glutamate carboxyl, in relation to the binuclear centre. Moreover, sequence comparisons reveal that several enzymes lacking that glutamate have a conserved substitution pattern in helix VI: -YSHPXV- instead of -XGHPEV-. These observations are discussed in terms of the mechanisms for proton uptake and it is suggested that, in these enzymes, tyrosine may play the role of the glutamate in the proton channel.

Structure analysis of cytochrome c3 from Desulfovibrio vulgaris Hildenborough at 1.9 A resolution.

The three-dimensional X-ray structure of cytochrome c3 from sulfate-reducing bacteria Desulfovibrio vulgaris Hildenborough (DvH) (M(r) 13 kDa, 107 residues, 4 heme groups) has been determined at 1.9 A resolution, by the method of molecular replacement, using the homologous part of the refined structure of cytochrome c3 from D. vulgaris Miyazaki F (DvMF). Crystals of c3 DvH were obtained with space group P61, a = 77.0 A, c = 77.2 A, Z = 12, corresponding to two independent molecules in the asymmetric unit. The structure was refined to an R-factor of 19.6%. The structures of the two molecules are analyzed, compared with each other and also with that of c3 DvMF. The main-chain atoms are, for the three structures, generally within 1.0 A. The intramolecular heme edge to edge distances and interplanar angles indicate two groups of values. Shorter distances are associated with near-normal angles, while longer distances with acute angles. Moreover, two of the four hemes, II and IV, are close to only one other heme, while the remaining two hemes, I and III, have two close neighbors each. The two histidine residues that co-ordinate the heme irons on the fifth and sixth positions are nearly parallel, except in the case of heme II. The only substitution from DvMF which is inside the molecule, A68V, occurs in the vicinity of that same heme. However, the non-paralellism between the two flanking histidine residues was also observed in DvMF. Heme II has a conserved higher exposure to solvent and one of the lowest redox potentials in the fully oxidized forms of the two cytochromes. A comparison between data obtained by spectroscopic techniques, nuclear magnetic resonance and electron paramagnetic resonance, and the structural results presented here, indicates two types of interactions, between hemes I and II and between hemes III and IV.

Crystal structure of desulforedoxin from Desulfovibrio gigas determined at 1.8 A resolution: a novel non-heme iron protein structure.

The crystal structure of desulforedoxin from Desulfovibrio gigas, a new homo-dimeric (2 x 36 amino acids) non-heme iron protein, has been solved by the SIRAS method using the indium-substituted protein as the single derivative. The structure was refined to a crystallographic R-factor of 16.9% at 1.8 A resolution. Native desulforedoxin crystals were grown from either PEG 4K or lithium sulfate, with cell constants a = b = 42.18 A, c = 72.22 A (for crystals grown from PEG 4K), and they belong to space group P3(2)21. The indium-substituted protein crystallized isomorphously under the same conditions. The 2-fold symmetric dimer is firmly hydrogen bonded and folds as an incomplete beta-barrel with the two iron centers placed on opposite poles of the molecule. Each iron atom is coordinated to four cysteinyl residues in a distorted tetrahedral arrangement. Both iron atoms are 16 A apart but connected across the 2-fold axis by 14 covalent bonds along the polypeptide chain plus two hydrogen bonds. Desulforedoxin and rubredoxin share some structural features but show significant differences in terms of metal environment and water structure, which account for the known spectroscopic differences between rubredoxin and desulforedoxin.

Crystallization and preliminary X-ray diffraction studies of a catechol-O-methyltransferase/inhibitor complex.

Inhibitors of the enzyme catechol-O-methyltransferase (COMT) are used as co-adjuvants in the therapy of Parkinson's disease. A recombinant form of the soluble cytosolic COMT from rat has been co-crystallized with a new potent inhibitor, BIA 8-176 [(3,4-dihydroxy-2-nitrophenyl)phenylmethanone], by the vapour-diffusion method using PEG 6K as precipitant. Crystals diffract to 1.6 A resolution on a synchrotron-radiation source and belong to the monoclinic space group P2(1), with unit-cell parameters a = 52.77, b = 79.63, c = 61.54 A, beta = 91.14 degrees.

Cytochrome c3 from Desulfovibrio gigas: crystal structure at 1.8 A resolution and evidence for a specific calcium-binding site.

Crystals of the tetraheme cytochrome c3 from sulfate-reducing bacteria Desulfovibrio gigas (Dg) (MW 13 kDa, 111 residues, four heme groups) were obtained and X-ray diffraction data collected to 1.8 A resolution. The structure was solved by the method of molecular replacement and the resulting model refined to a conventional R-factor of 14.9%. The three-dimensional structure shows many similarities to other known crystal structures of tetraheme c3 cytochromes, but it also shows some remarkable differences. In particular, the location of the aromatic residues around the heme groups, which may play a fundamental role in the electron transfer processes of the molecule, are well conserved in the cases of hemes I, III, and IV. However, heme II has an aromatic environment that is completely different to that found in other related cytochromes c3. Another unusual feature is the presence of a Ca2+ ion coordinated by oxygen atoms supplied by the protein within a loop near the N-terminus. It is speculated that this loop may be stabilized by the presence of this Ca2+ ion, may contribute to heme-redox perturbation, and might even be involved in the specificity of recognition with its redox partner.

Preliminary crystallographic analysis of the oxidized form of a two mono-nuclear iron centres protein from Desulfovibrio desulfuricans ATCC 27774.

Crystals of the fully oxidized form of desulfoferrodoxin were obtained by vapor diffusion from a solution containing 20% PEG 4000, 0.1 M HEPES buffer, pH 7.5, and 0.2 M CaCl2. Trigonal and/or rectangular prisms could be obtained, depending on the temperature used for the crystal growth. Trigonal prisms belong to the rhombohedral space group R32, with a = 112.5 A and c = 63.2 A; rectangular prisms belong to the monoclinic space group C2, with a = 77.7 A, b = 80.9 A, c = 53.9 A, and beta = 98.1 degrees. The crystallographic asymmetric unit of the rhombohedral crystal form contains one molecule. There are two molecules in the asymmetric unit of the monoclinic form, in agreement with the self-rotation function.

Synthesis, anticonvulsant properties and pharmacokinetic profile of novel 10,11-dihydro-10-oxo-5H-dibenz/b,f/azepine-5-carboxamide derivatives.

A series of novel derivatives of oxcarbazepine (5), 10,11-dihydro-10-oxo-5H-dibenz/b,f/azepine-5-carboxamide was synthesised and evaluated for their anticonvulsant activity and sodium channel blocking properties. The oxime 8 was found to be the most active compound from this series, displaying greater potency than its geometric isomer 9 and exhibiting also the highest protective index value. Importantly, the metabolic profile of 8 differs from the already established dibenz/b,f/azepine-5-carboxamide drugs such as 1 and 5 which undergo rapid and complete conversion in vivo to several biologically active metabolites. In contrast 8 is metabolised to only a very minor extent leading to the conclusion that the observed anti-convulsant effect is solely attributable to 8. It is concluded that 8 may be as effective as 1 and 5 at controlling seizures and that the low toxicity and consequently high protective index should provide the compound with an improved side-effect profile.

Comparative study of ortho- and meta-nitrated inhibitors of catechol-O-methyltransferase: interactions with the active site and regioselectivity of O-methylation.

In this work, we present a comparative case study of "ortho-" and "meta-nitrated" catecholic inhibitors of catechol-O-methyltransferase (COMT), with regard to their interaction with the catalytic site of the enzyme and the in vitro regioselective formation of their mono-O-methyl ether metabolites. In particular, the effects of altering the attachment position of the inhibitors' side-chain substituent, within the classic nitrocatechol pharmacophore, were investigated. For this purpose, we compared two simple regioisomeric nitrocatechol-type inhibitors of COMT, BIA 3-228 and BIA 8-176, which contain the benzoyl substituent attached at the meta and ortho positions, respectively, relative to the nitro group. The two compounds were slowly O-methylated by COMT in vitro, but the particular substitution pattern of each compound was shown to have a profound impact on the regioselectivity of their O-methylation. To provide a plausible interpretation of these results, a comprehensive analysis of the protein-inhibitor interactions and of the relative chemical susceptibility to O-methylation of the catechol hydroxyl groups was performed by means of docking simulations and ab initio molecular orbital calculations. The major structural and chemical factors that determine the enzyme regioselectivity of O-methylation were identified, and the X-ray structure of the complex of COMT with S-adenosyl-l-methionine and BIA 8-176 is herein disclosed. This is the first reported structure of the soluble form of COMT complexed with a nitrocatecholic inhibitor having a bulky substituent group in adjacent position (ortho) to the nitro group. Structural and dynamic aspects of this complex are analyzed and discussed, in the context of the present study.

Improvement of side-chain modeling in proteins with the self-consistent mean field theory method based on an analysis of the factors influencing prediction.

With the objective of improving side-chain conformation prediction, we have analyzed the influence of various factors on prediction by the Self-Consistent Mean Field Theory method, applied to a set of high resolution x-ray protein structure models. These factors may be classed as variations in the mean field optimization protocol, variations in the potential energy function, and variations in rotamer library completeness. We have developed an optimization protocol that consistently reached lower mean field conformational free energies than two other protocols. This protocol led to an important improvement in prediction. We observed a major improvement in prediction with two more detailed van der Waals parameter sets, which we found to be due mainly to the introduction of scaling of 1-4 interactions. In a comparison of two knowledge-based rotamer libraries of considerably different size, we observed an unexpected decrease in prediction with an increase in library completeness. However, when we introduced a torsion potential term in the potential energy function, we found an important increase in average prediction and in the prediction of almost all residue types with a more complete rotamer set. The two knowledge-based rotamer libraries now became equivalent in terms of average prediction. The results we obtained in an analysis of the effect of the introduction of an additional electrostatic term in the potential energy function were largely inconclusive. However, we found a small increase in average prediction for an electrostatic potential term with a fixed dielectric constant of 15. The combined effect of all the factors we analyzed in this study resulted in average prediction accuracies of 79.9% for X1, 68.1% for X1 + 2, and 1.590 A for global rms deviation (RMSD); the corresponding values for core residues were 88.2%, 78.6%, and 1.171 A. These values represent improvements in average prediction of 6.5% for X1, 9.1% for X1 + 2, and 0.163 A for global RMSD over the original conditions; the corresponding improvements in the core were 5.9%, 9.0%, and 0.180 A, respectively.

Ab initio structure solution of a dimeric cytochrome c3 from Desulfovibrio gigas containing disulfide bridges.

The 1.2 A resolution crystal structure of the 29 kDa di-tetrahaem cytochrome c3 from the sulfate reducing bacterium Desulfovibrio gigas was solved by ab initio methods, making this the largest molecule to be solved by this procedure. The actual refined model of the cysteine-linked dimeric molecule reveals that this molecule is very similar to the non-covalently linked symmetrical dimer of the di-tetrahaem cytochrome c3 from Desulfomicrobium norvegicum. Each monomer has the typical polypeptide fold, haem arrangement and iron coordination found for the tetrahaem cytochrome c3 molecules. The interface between the covalently linked monomers in the asymmetric unit of the crystal shows a pseudo two-fold arrangement, disturbed from symmetry by crystal packing forces. The fact that D. gigas contains a dimeric tetrahaem cytochrome with solvent accessible disulfide bridges and that this cytochrome specifically couples hydrogen oxidation to thiosulfate reduction in bacterial extracts provides an interesting aspect related to disulfide exchange reactions in this microorganism.

Crystallization and preliminary X-ray diffraction analysis of tetra-heme cytochrome c3 from sulfate- and nitrate-reducing Desulfovibrio desulfuricans ATCC 27774.

Crystals of the tetra-heme cytochrome c(3) (M(r) = 13 kDa, 107 residues, four heme groups) from sulfate- and nitrate-reducing Desulfovibrio desulfuricans ATCC 27774 have been obtained and crystallographically characterized. They belong to space group P6(1)22 with cell dimensions a = b = 61.84 (4) and c = 109.7 (2) A, and Z = 12. Intensity data were initially collected on a FAST system with a rotating-anode X-ray source leading to a total of 22 592 observations, from which only 4930 were unique, in the resolution range 20.0-2.4 A with an R(merge)(I) of 7.0%. Higher resolution data were measured on a FAST system at station 9.6 of the SRS (Daresbury, England), leading to 19 328 intensities, of which 11 179 were unique, in the resolution range 20.0-1.75 A and an R(merge)(I) of 5.5%. Cross-rotation and translation functions were performed with ALMN and TFSGEN programs from the CCP4 suite. The packing of the molecules in the unit cell was checked with TOM/FRODO. Rigid-body refinement of the model and subsequent refinement using molecular dynamics were performed with X-PLOR, leading to a current R factor of 25.9%, for data up to 2.3 A.

Implicit solvation in the self-consistent mean field theory method: sidechain modelling and prediction of folding free energies of protein mutants.

The Atomic Solvation Parameter (ASP) model is one of the simplest models of solvation, in which the solvation free energy of a molecule is proportional to the solvent accessible surface area (SAS) of its atoms. However, until now this model had not been incorporated into the Self-Consistent Mean Field Theory (SCMFT) method for modelling sidechain conformations in proteins. The reason for this is that SAS is a many-body quantity and, thus, it is not obvious how to define it within the Mean Field (MF) framework, where multiple copies of each sidechain exist simultaneously. Here, we present a method for incorporating an SAS-based potential, such as the ASP model, into SCMFT. The theory on which the method is based is exact within the MF framework, that is, it does not depend on a pairwise or any other approximation of SAS. Therefore, SAS can be calculated to arbitrary accuracy. The method is computationally very efficient: only 7.6% slower on average than the method without solvation. We applied the method to the prediction of sidechain conformation, using as a test set high-quality solution structures of 11 proteins. Solvation was found to substantially improve the prediction accuracy of well-defined surface sidechains. We also investigated whether the methodology can be applied to prediction of folding free energies of protein mutants, using a set of barnase mutants. For apolar mutants, the modest correlation observed between calculated and observed folding free energies without solvation improved substantially when solvation was included, allowing the prediction of trends in the folding free energies of this type of mutants. For polar mutants, correlation was not significant even with solvation. Several other factors also responsible for the correlation were identified and analysed. From this analysis, future directions for applying and improving the present methodology are discussed.