[Topical therapy of inflammatory dermatoses, pruritus and pain, as well as hyperhidrosis]. / Topische Therapie von entzündlichen Dermatosen, Juckreiz und Schmerz sowie Hyperhidrose.

In this review, substances suitable for the topical treatment of inflammatory dermatoses, pruritus, pain and hyperhidrosis are presented. Both non-specific and specific topical treatments are discussed with respect to their mode of action, indications and side effects; included are corticosteroids, calcineurin inhibitors, dithranol, coal tar, liquor carbonis detergens, vitamin D analogues, metronidazole, azelaic acid, retinoids, and benzoyl peroxide. Additionally, practical directions for the treatment of these common skin diseases are provided.

[Topical treatment of infections, tumors and hyperkeratotic disorders]. / Topische Therapie von Infektionen, Hauttumoren und Hyperkeratosen.

In this review substances for the topical treatment of skin infections, skin tumors and hyperkeratotic disorders are presented. Substances with specific actions as topical antimycotic, antibiotic, antiseptic, antiviral and antiparasitic agents as well as diclofenac in hyaluronic acid, 5-fluoruracil, imiquimod, ingenol mebutate, bexarotene, mechlorethamine, BCNU, alitretinoin, silicone gel, salicylic acid and urea are discussed with respect to their mode of action, indication and side effects.

[Cutaneous squamous cell carcinoma: a review with consideration of special patient groups]. / Kutanes Plattenepithelkarzinom unter Berücksichtigung besonderer Patientengruppen.

The incidence of non-melanoma skin cancer (NMSC) is increasing. Squamous cell carcinoma of the skin (SCC) is a tumor of the elderly. Due to the increasing life expectancy, SCC will become more and more frequent in the future. Generally SCC has a favorable prognosis. Standard therapy is microscopically- controlled excision. Therapy of advanced and metastatic SCC is still challenging. Patients with regional lymph node metastasis have ten-year survival rates less than 20%; patients with distant metastases less than 10%. Immunosuppression has been shown to be one of the key prognostic factors for metastasis. The article reviews SCC and focusses on patients being at risk for an unfavorable course.

Effect of antenatal corticosteroid treatment on the fetal lung: a magnetic resonance imaging study.

OBJECTIVES: To investigate the effect of antenatal corticosteroid treatment on the fetal lung using magnetic resonance imaging. METHODS: Prospective evaluation of 30 consecutive singleton pregnancies that received antenatal corticosteroid treatment (12 mg betamethasone i.m. on admission and 24 h later) because of threatened preterm birth. Fetal lungs were assessed using T2-weighted single-shot fast spin-echo images of a whole-body 1.5-T superconducting unit twice: less than 24 h and more than 48 h after the first course of betamethasone. Lung volumes and lung-liver signal-intensity ratios were compared between the two time points. RESULTS: Nine patients had to be excluded from the analysis because they did not complete the study protocol as required. Ten female and 11 male fetuses with a gestational age between 23.4 and 32.6 weeks were included in the final analysis. The mean gestational age of included fetuses was 27.5 ± 2.8 weeks. Using a linear regression model, a significant influence of gestational age on ln fetal lung volume (r(2)=0.414; P<0.0001) and lung-liver signal-intensity ratios (r(2)=0.271, P<0.0001) was found. Between the two evaluated time points, a significant increase in lung-liver signal-intensity ratios (2.34 ± 0.72 vs. 3.22 ± 1.12, P<0.0001), but not in mean lung volumes (46.6 ± 20.7 cm(3) vs. 48.8 ± 16.0 cm(3) , P=0.292), was observed. CONCLUSION: We demonstrate a significant increase in lung-liver signal-intensity ratios after antenatal corticosteroid treatment for induction of lung maturation which most likely reflects changing properties of the fetal lung parenchyma. This could potentially be useful in non-invasively assessing the effect of antenatal corticosteroid treatment on the lungs of fetuses at risk for preterm birth.

Magnetic resonance imaging of the placenta identifies placental vascular abnormalities independently of Doppler ultrasound.

OBJECTIVE: To evaluate the relationship between placental vascular pathology detected by prenatal magnetic resonance imaging (MRI) and perinatal outcome. METHODS: This was a retrospective, hospital-based, cross-sectional study in which all fetal MRI examinations of singleton pregnancies with vascular placental pathology (i.e. infarction with/without hemorrhage, subchorionic thrombi/hemorrhages, intervillous thrombi/hemorrhages, or retroplacental hematoma) in the period 2002-2007 were included. The extent of the pathology was expressed as a percentage of the total placental volume. Abnormalities of umbilical artery Doppler ultrasound examinations within 7 days between MRI and ultrasound examination were noted. Death in utero or postnatally was the primary outcome. Gestational age at MRI and at birth and the occurrence of intrauterine growth restriction (IUGR) were noted. Logistic regression analysis was performed to assess the impact of gestational age at MRI, extent of the vascular lesion and presence of pathological Doppler ultrasound measurements on the prediction of mortality. RESULTS: Fifty-nine structurally normal singleton pregnancies with placental vascular abnormalities were included in the analysis. Mortality rate was 36%; among the survivors, 87% were born before 37 + 0 gestational weeks and 50% suffered from IUGR. In 55% of the pregnancies pathological umbilical artery Doppler findings were identified, of which 27% were non-survivors. Mortality was predicted by earlier gestational age at fetal MRI for placental pathology (P < 0.05) and increasing extent of the vascular lesion (P < 0.05), but not by the presence of pathological Doppler ultrasound data. Accuracy of the prediction was 82%, sensitivity was 67% and specificity 89%. CONCLUSION: MRI-detected vascular placental pathologies may help to identify pregnancies at risk for adverse outcome and fetal death independently of umbilical artery Doppler status.

Glutaconate CoA-transferase from Acidaminococcus fermentans: the crystal structure reveals homology with other CoA-transferases.

BACKGROUND: Coenzyme A-transferases are a family of enzymes with a diverse substrate specificity and subunit composition. Members of this group of enzymes are found in anaerobic fermenting bacteria, aerobic bacteria and in the mitochondria of humans and other mammals, but so far none have been crystallized. A defect in the human gene encoding succinyl-CoA: 3-oxoacid CoA-transferase causes a metabolic disease which leads to severe ketoacidosis, thus reflecting the importance of this family of enzymes. All CoA-transferases share a common mechanism in which the CoA moiety is transferred from a donor (e.g. acetyl CoA) to an acceptor, (R)-2-hydroxyglutarate, whereby acetate is formed. The transfer has been described by a ping-pong mechanism in which CoA is bound to the active-site residue of the enzyme as a covalent thiol ester intermediate. We describe here the crystal structure of glutaconate CoA-transferase (GCT) from the strictly anaerobic bacterium Acidaminococcus fermentans. This enzyme activates (R)-2-hydroxyglutarate to (R)-2-hydroxyglutaryl-CoA in the pathway of glutamate fermentation. We initiated this project to gain further insight into the function of this enzyme and the structural basis for the characteristics of CoA-transferases. RESULTS: The crystal structure of GCT was solved by multiple isomorphous replacement to 2.55 A resolution. The enzyme is a heterooctamer and its overall arrangement of subunits can be regarded as an (AB)4tetramer obeying 222 symmetry. Both subunits A and B belong to the open alpha/beta-protein class and can be described as a four-layered alpha/alpha/beta/alpha type with a novel composition and connectivity of the secondary structure elements. The core of subunit A consists of seven alpha/beta repeats resulting in an all parallel central beta sheet, against which helices pack from both sides. In contrast, the centre of subunit B is formed by a ninefold mixed beta sheet. In both subunits the helical C terminus is folded back onto the N-terminal domain to form the third layer of helices. CONCLUSIONS: The active site of GCT is located at the interface of subunits A and B and is formed by loops of both subunits. The funnel-shaped opening to the active site has a depth and diameter of about 20 A with the catalytic residue, Glu54 of subunit B, at the bottom. The active-site glutamate residue is stabilized by hydrogen bonds. Despite very low amino acid sequence similarity, subunits A and B reveal a similar overall fold. Large parts of their structures can be spatially superimposed, suggesting that both subunits have evolved from a common ancestor.

X-ray structures and mechanistic implications of three functional derivatives of ascorbate oxidase from zucchini. Reduced, peroxide and azide forms.

The X-ray structures of three functional derivatives of ascorbate oxidase (EC 1.10.3.3) from Zucchini have been determined and are compared to the "native" oxidized form. The fully reduced form of ascorbate oxidase has been refined to a crystallographic R-factor of 19.6% for all reflections between 8.0 A and 2.2 A resolution. The geometry at the type-1 copper (CU1) is unchanged compared to the oxidized form, but the oxygen ligand bridging the copper ions CU2 and CU3 (spectroscopic type-3 copper pair) is released and the copper ions move apart yielding a trigonal planar co-ordination with their ligating histidine residues. The co-ordination at the copper ion CU4 (spectroscopic type-2 copper) is not affected. The copper-copper distances increase from an average 3.7 A in the native form to 5.1 A for CU2-CU3, 4.4 A for CU2-CU4 and 4.1 A for CU3-CU4. The peroxide derivative of ascorbate oxidase has been refined to a crystallographic R-factor of 16.0% for all reflections between 8.0 A and 2.59 A resolution. The geometry at the type-1 copper site is not changed compared to the oxidized form. The oxygen ligand bridging copper atoms CU2 and CU3 is lost, too. The peroxide binds terminally to the copper ion CU2 as hydroperoxide. Copper ion CU2 is fourfold co-ordinated to the NE2 atoms of the three histidine residues and to the oxygen atom of the terminally bound peroxide molecule in a distorted tetrahedral geometry. Copper ion CU3 is threefold co-ordinated as in the reduced form and co-ordination around copper atom CU4 is unaltered. The copper-copper distances increase to 4.8 A for CU2-CU3 and 4.5 A for CU2-CU4. The distance CU3-CU4 remains 3.7 A. Treatment with peroxide causes a partial depletion of copper ion CU2. The refinement for the azide derivative of ascorbate oxidase converged at a crystallographic R-factor of 17.8% for all reflections between 8.0 A and 2.32 A. There are no significant structural changes at the type-1 copper site. The oxygen ligand bridging copper ions CU2 and CU3 is again released. Two azide molecules bind terminally to copper ion CU2. Copper ion CU2 is fivefold co-ordinated to the NE2 atoms of the three histidine residues and to both terminally bound azide molecules in a trigonal-bipyramidal manner. Copper-copper distances increase to 5.1 A for CU2-CU3 and 4.6 A for CU2-CU4. The distance CU3-CU4 is decreased to 3.6 A.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystal structures of cystathionine gamma-synthase inhibitor complexes rationalize the increased affinity of a novel inhibitor.

Cystathionine gamma-synthase catalyzes the committed step of methionine biosynthesis. This pathway is unique to microorganisms and plants, rendering the enzyme an attractive target for the development of antimicrobials and herbicides. We solved the crystal structures of complexes of cystathionine gamma-synthase (CGS) from Nicotiana tabacum with inhibitors of different compound classes. The complex with the substrate analog dl-E-2-amino-5-phosphono-3-pentenoic acid verifies the carboxylate-binding function of Arg423 and identifies the phosphate-binding pocket of the active site. The structure shows the function of Lys165 in specificity determination and suggests a role for the flexible side-chain of Tyr163 in catalysis. The importance of hydrophobic interactions for binding to the active-site center is highlighted by the complex with 3-(phosphonomethyl)pyridine-2-carboxylic acid. The low affinity of this compound is due to the non-optimal arrangement of the functional groups binding to the phosphate and carboxylate-recognition site, respectively. The newly identified inhibitor 5-carboxymethylthio-3-(3'-chlorophenyl)-1,2,4-oxadiazol, in contrast, shows the highest affinity to CGS reported so far. This affinity is due to binding to an additional active-site pocket not used by the physiological substrates. The inhibitor binds to the carboxylate-recognition site, and its tightly bent conformation enables it to occupy the novel binding pocket between Arg423 and Ser388. The described structures suggest improvements for known inhibitors and give guidelines for the development of new lead compounds.

Crystal structure of the pyridoxal-5'-phosphate dependent cystathionine beta-lyase from Escherichia coli at 1.83 A.

Cystathionine beta-lyase (CBL) is a member of the gamma-family of PLP-dependent enzymes, that cleaves C beta-S bonds of a broad variety of substrates. The crystal structure of CBL from E. coli has been solved using MIR phases in combination with density modification. The structure has been refined to an R-factor of 15.2% at 1.83 A resolution using synchroton radiation diffraction data. The asymmetric unit of the crystal cell (space group C222(1)) contains two monomers related by 2-fold symmetry. A homotetramer with 222 symmetry is built up by crystallographic and non-crystallographic symmetry. Each monomer of CBL can be described in terms of three spatially and functionally different domains. The N-terminal domain (residues 1 to 60) consists of three alpha-helices and one beta-strand. It contributes to tetramer formation and is part of the active site of the adjacent subunit. The second domain (residues 61 to 256) harbors PLP and has an alpha/beta-structure with a seven-stranded beta-sheet as the central part. The remaining C-terminal domain (residues 257 to 395), connected by a long alpha-helix to the PLP-binding domain, consists of four helices packed on the solvent-accessible side of an antiparallel four-stranded beta-sheet. The fold of the C-terminal and the PLP-binding domain and the location of the active site are similar to aminotransferases. Most of the residues in the active site are strongly conserved among the enzymes of the transsulfuration pathway. Additionally, CBL is homologous to the mal gamma gene product indicating an evolutionary relationship between alpha and gamma-family of PLP-dependent enzymes. The structure of the beta, beta, beta-trifluoroalanine inactivated CBL has been refined at 2.3 A resolution to an R-factor of 16.2%. It suggests that Lys210, the PLP-binding residue, mediates the proton transfer between C alpha and S gamma.

X-ray analysis and spectroscopic characterization of M121Q azurin. A copper site model for stellacyanin.

The dependence of the properties of the azurin blue copper site on the nature of the axial ligand at position 121 was tested by site-directed mutagenesis. This residue was substituted for a glutamine, the purported fourth copper ligand in the related protein stellacyanin. M121Q azurin was isolated and purified from Escherichia coli and characterized by spectroscopic methods. The mutant copper site has the ultra-violet-vis and electron paramagnetic resonance (EPR) characteristics of a type I site, but the spectroscopic details differ significantly from wild-type (wt) azurin. The X and S-band EPR spectra of M121Q azurin can be well stimulated with the parameters for stellacyanin, indicating that the copper sites of both proteins in the oxidized state are similar. The midpoint potential of M121Q is 263 mV, 25 mV lower than for wt azurin. The reactivity of the mutant was probed by measuring the electron self exchange rate by nuclear magnetic resonance spectroscopy. The rate was 8 x 10(3) mol-1 s-1, almost two orders of magnitude lower than the value for wt azurin (5 x 10(5) mol-1 s-1). Detailed structural information on the M121Q Cu(II)-site was obtained by X-ray analysis of M121Q azurin crystals at 1.9 A resolution. The histidine and cysteine copper ligand distances and angles in the equatorial plane around the copper are very similar to the wt protein. Gln121 is co-ordinated in a monodentate fashion via its side-chain oxygen atom at a distance of 2.26 A. The distance between copper and the carbonyl group of Gly45 is increased from 3.13 A (wt) to 3.37 A resulting in a distorted tetrahedral N2SO copper co-ordination. The possible significance of these results for the structure of the copper site of stellacyanin, the only small blue copper protein lacking a methionine ligand, is discussed. Conformational changes with respect to the wt azurin are seen in some of the connecting loops between secondary structure elements, in the mutation site and in the beta-strand 2a. The side-chains involved in the hydrophobic patch surrounding His117 are subject to large changes in their conformations. In contrast to wt azurin, the copper site in M121Q azurin undergoes significant structural changes on reduction.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystallization, crystal structure analysis and preliminary molecular model of the bilin binding protein from the insect Pieris brassicae.

The bilin binding protein of the butterfly Pieris brassicae has been prepared, crystallized and its crystal structure determined at high resolution using film and FAST area detector intensity data. The crystallographic asymmetric unit contains a tetramer of identical subunits with a molecular weight of about 90,000. The crystal structure was determined by isomorphous replacement. Use was made of the molecular symmetry to improve phases. A molecular interpretation of the electron density distribution and partial tracing of the polypeptide chain was possible without amino acid sequence information, as the fold is very similar to retinol binding protein. It is characterized by a beta-barrel formed by two orthogonal beta-sheets and an alpha-helix. The bilin pigment seems to be bound within the beta-barrel analogously to retinol in retinol binding protein. The tetramer in the crystal has C2 symmetry and is a dimer of dimers of quasi-equivalent subunits.

Refined crystal structure of ascorbate oxidase at 1.9 A resolution.

The crystal structure of the fully oxidized form of ascorbate oxidase (EC 1.10.3.3) from Zucchini has been refined at 1.90 A (1 A = 0.1 nm) resolution, using an energy-restrained least-squares refinement procedure. The refined model, which includes 8764 protein atoms, 9 copper atoms and 970 solvent molecules, has a crystallographic R-factor of 20.3% for 85,252 reflections between 8 and 1.90 A resolution. The root-mean-square deviation in bond lengths and bond angles from ideal values is 0.011 A and 2.99 degrees, respectively. The subunits of 552 residues (70,000 Mr) are arranged as tetramers with D2 symmetry. One of the dyads is realized by the crystallographic axis parallel to the c-axis giving one dimer in the asymmetric unit. The dimer related about this crystallographic axis is suggested as the dimer present in solution. Asn92 is the attachment site for one of the two N-linked sugar moieties, which has defined electron density for the N-linked N-acetyl-glucosamine ring. Each subunit is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type and related to plastocyanin and azurin. An analysis of intra- and intertetramer hydrogen bond and van der Waals interactions is presented. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine and a methionine ligand and represents the type-1 copper. It is located in domain 3. The bond lengths of the type-1 copper centre are comparable to the values for oxidized plastocyanin. The trinuclear cluster has eight histidine ligands symmetrically supplied from domain 1 and 3. It may be subdivided into a pair of copper atoms with histidine ligands whose ligating N-atoms (5 NE2 atoms and one ND1 atom) are arranged trigonal prismatic. The pair is the putative type-3 copper. The remaining copper has two histidine ligands and is the putative spectroscopic type-2 copper. Two oxygen atoms are bound to the trinuclear species as OH- or O2- and bridging the putative type-3 copper pair and as OH- or H2O bound to the putative type-2 copper trans to the copper pair. The bond lengths within the trinuclear copper site are similar to comparable binuclear model compounds. The putative binding site for the reducing substrate is close to the type-1 copper.(ABSTRACT TRUNCATED AT 400 WORDS)

Enzymatic mechanism of creatine amidinohydrolase as deduced from crystal structures.

Crystal structures of the enzyme creatine amidinohydrolase (creatinase, EC 3.5.3.3) with two different inhibitors, the reaction product sarcosine and the substrate creatine, bound have been analyzed by X-ray diffraction methods. With the inhibitor carbamoyl sarcosine, two different crystal forms at different pH values have been determined. An enzymatic mechanism is proposed on the basis of the eight structures analyzed. The enzyme binds substrate and inhibitor in a distorted geometry where the urea resonance is broken. His232 is the general base and acid, and acts as a proton shuttle. It withdraws a proton from water 377 and donates it to the N(3) atom of the guanidinium group. OH- 377 adds to the C(1) atom of the guanidinium group to form a urea hydrate. Proton withdrawal by His232 leads to products. The reaction product sarcosine binds to the active site in a reverse orientation. The free enzyme was found to have a bicarbonate bound to the active site.

Crystal structure analysis of oxidized Pseudomonas aeruginosa azurin at pH 5.5 and pH 9.0. A pH-induced conformational transition involves a peptide bond flip.

The X-ray crystal structure of recombinant wild-type azurin from Pseudomonas aeruginosa was determined by difference Fourier techniques using phases derived from the structure of the mutant His35Leu. Two data sets were collected from a single crystal of oxidized azurin soaked in mother liquor buffered at pH 5.5 and pH 9.0, respectively. Both data sets extend to 1.93 A resolution. The two pH forms were refined independently to crystallographic R-factors of 17.6% (pH 5.5) and 17.5% (pH 9.0). The conformational transition previously attributed to the protonation/deprotonation of residue His35 (pKa(red) = 7.3, pKa(ox) = 6.2), which lies in a crevice of the protein close to the copper binding site, involves a concomitant Pro36-Gly37 main-chain peptide bond flip. At the lower pH, the protonated imidazole N delta 1 of His35 forms a strong hydrogen bond with the carbonyl oxygen from Pro36, while at alkaline pH the deprotonated N delta 1 acts as an acceptor of a weak hydrogen bond from HN Gly37. The structure of the remainder of the azurin molecule, including the copper binding site, is not significantly affected by this transition.

X-ray crystal structure of the two site-specific mutants His35Gln and His35Leu of azurin from Pseudomonas aeruginosa.

The three-dimensional structures of two site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa have been solved by a combination of isomorphous replacement and Patterson search techniques, and refined by energy-restrained least-squares methods. The mutations introduced by recombinant DNA techniques involve residue His35, which was exchanged for glutamine and leucine, to probe for its suggested role in electron transfer. The two mutants, His35Gln (H35Q) and His35Leu (H35L), crystallize non-isomorphously in the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 109.74 A, b = 99.15 A, c = 47.82 A for H35Q, a = 57.82 A, b = 81.06 A, c = 110.03 A for H35L. In each crystal form, there are four molecules in the asymmetric unit. They are arranged as a dimer of dimers in the H35Q case and are distorted from ideal C2 symmetry in H35L. The final crystallographic R-value is 16.3% for 20.747 reflections to a resolution of 2.1 A for H35Q and 17.0% for 32,548 reflections to 1.9 A for H35L. The crystal structures reported here represent the first crystallographically refined structures for azurin from P. aeruginosa. The structure is very similar to that of azurin from Alcaligenes denitrificans. The copper atom is located about 7 A below a hydrophobic surface region and is ligated by five donor groups in a distorted trigonal bipyramidal fashion. The implications for electron transfer properties of the protein are discussed in terms of the mutation site and the packing of the molecules within the tetramer.

Ta6Br(2+)12, a tool for phase determination of large biological assemblies by X-ray crystallography.

The title compound Ta6Br(2+)12 is of interest for the analysis of biological structures as a heavy-metal derivative with great potential for the structure determination of large protein systems. In macromolecular crystallography the phases of the measured structure factor amplitudes have to be determined. The most widely used method for novel structures is isomorphous replacement by introducing electron-rich compounds into the protein crystals. These compounds produce measurable changes of the diffraction intensities, which allow phase determination. We synthetized the Ta6Br(2+)12 cluster in high yields, crystallized it, and determined its crystal structure by X-ray diffraction analysis at atomic resolution. The cluster is a regular octahedron consisting of six metal atoms with 12 bridging bromine atoms along the 12 edges of the octahedron. The cluster is compact, of approximately spherical shape with about 4.3 A radius and highly symmetrical. One Ta6Br(2+)12 ion adds 856 electrons to a protein, a considerable contribution to the scattering power even of large proteins or multimeric systems. At low resolution all atoms of the cluster scatter in phase and act as a super heavy-atom, which is easy to locate in the difference Patterson map. We investigated its binding sites in the biologically significant high-resolution structures of an antibody V(L) domain, dimethyl sulfoxide reductase, GTP-cyclohydrolase I, and the proteasome. With the randomly oriented cluster, treated as a single site scatterer, phases could be used only up to 6 A resolution. In contrast, when the cluster is correctly oriented, phases calculated from its 18 atom sites can be used to high resolution. We present the atomic structure of the Ta6Br(2+)12, describe a method to determine its localization and orientation in the unit cell of protein crystals of two different proteins, and analyse its phasing power. We show that phases can be calculated to high resolution. The phase error is lower by more than 30 degrees compared to the single site approximation, using a resolution of 2.2 A. Furthermore, Ta6Br(2+)12 has two different strong anomalous scatterers tantalum and bromine to be used for phase determination.

X-ray crystal structure of the two site-specific mutants Ile7Ser and Phe110Ser of azurin from Pseudomonas aeruginosa.

The blue copper protein azurin from Pseudomonas aeruginosa contains a single Trp residue that is believed to be involved in the inducible intramolecular electron transfer from a disulphide group to the copper centre. This residue shows in fluorescence spectra the highest energy emission of tryptophan-containing compounds at room temperature, which is explained by its rigid and highly hydrophobic environment. In order to investigate the role of the Trp residue in electron transfer and the influence of its environment, two mutations (17S and F110S) were introduced that were thought to increase the polarity and the mobility in its environment. The crystal structures of these mutants were solved at 2.2 A and 2.3 A resolution, respectively. These provide a structural basis for the changes observed in fluorescence spectra compared with the wild-type protein. We conclude from our results that these changes are not caused by a change in the dynamics of the Trp residue itself, but exclusively by an increased effective dielectric constant of the microenvironment of Trp48 and by changes in mobility of the mutated residues.

The crystal structure of cystathionine gamma-synthase from Nicotiana tabacum reveals its substrate and reaction specificity.

Cystathionine gamma-synthase catalyses the committed step of de novo methionine biosynthesis in micro-organisms and plants, making the enzyme an attractive target for the design of new antibiotics and herbicides. The crystal structure of cystathionine gamma-synthase from Nicotiana tabacum has been solved by Patterson search techniques using the structure of Escherichia coli cystathionine gamma-synthase. The model was refined at 2.9 A resolution to a crystallographic R -factor of 20.1 % (Rfree25.0 %). The physiological substrates of the enzyme, L-homoserine phosphate and L-cysteine, were modelled into the unliganded structure. These complexes support the proposed ping-pong mechanism for catalysis and illustrate the dissimilar substrate specificities of bacterial and plant cystathionine gamma-synthases on a molecular level. The main difference arises from the binding modes of the distal substrate groups (O -acetyl/succinyl versusO -phosphate). Central in fixing the distal phosphate of the plant CGS substrate is an exposed lysine residue that is strictly conserved in plant cystathionine gamma-synthases whereas bacterial enzymes carry a glycine residue at this position. General insight regarding the reaction specificity of transsulphuration enzymes is gained by the comparison to cystathionine beta-lyase from E. coli, indicating the mechanistic importance of a second substrate binding site for L-cysteine which leads to different chemical reaction types.

Crystal structure analysis and refinement at 2.15 A resolution of amicyanin, a type I blue copper protein, from Thiobacillus versutus.

The crystal structure of the type I blue copper protein amicyanin from Thiobacillus versutus has been determined by Patterson search techniques on the basis of the molecular model of amicyanin from Paracoccus denitrificans, and refined by energy-restrained least-squares methods. Amicyanin crystallizes in the trigonal space group P3(2) with unit cell dimensions of a = b = 87.40 A, c = 38.20 A. The asymmetric unit is composed of three independent molecules centred on the crystallographic 3(2) axes. The final R-value is 17.4% for 15,984 reflections to a resolution of 2.15 A. The polypeptide fold in amicyanin is based on the beta-sandwich structure commonly found in blue copper proteins. Nine beta strands are folded into two twisted beta-sheets that pack together with a filling of non-polar residues between them. The geometry of the copper site is similar to that of plastocyanin. There are four ligands, arranged approximately as a distorted tetrahedron, to the copper atom: His54, Cys93, His96 and Met99. One of the copper ligands, His96, is exposed to the surface and lies in the centre of a cluster of seven hydrophobic residues.

X-ray crystal structure of the blue oxidase ascorbate oxidase from zucchini. Analysis of the polypeptide fold and a model of the copper sites and ligands.

Two crystal forms of the multi-copper protein ascorbate oxidase from Zucchini have been analysed at 2.5 A (1 A = 0.1 nm) resolution and a model of the polypeptide chain and the copper ions and their ligands has been built. Crystal forms M2 and M1 contain a dimer of 140,000 Mr and a tetramer of 280,000 Mr, respectively, in the asymmetric unit. The crystallographic analysis proceeded by multiple isomorphous replacement in M2 followed by solvent flattening and averaging about the local dyad axis. M1 was solved by Patterson search techniques using the M2 electron density. M1 was fourfold averaged. M1 and M2 were combined and the process of averaging repeated in cycles. An atomic model was built into the resulting electron density map and refinement initiated. The current R values of M2 and M1 are 24.5% and 32.6%, respectively. Excellent stereo chemistry was maintained, with root-mean-square deviations of bond lengths and bond angles from average values of 0.02 A and 3.1 degrees, respectively. Each subunit of about 550 amino acid residues has a globular shape with dimensions of 49 A x 53 A x 65 A. It is built up by three domains arranged sequentially on the polypeptide chain and tightly associated in space. The folding of all three domains is of a similar beta-barrel type. It is distantly related to plastocyanin. Each subunit has four copper atoms bound as mononuclear and trinuclear species. The mononuclear copper has two histidine, a cysteine, and a methionine ligand and represents the type-1 copper. It is located in the third domain. The trinuclear cluster has eight histidine ligands. It may be subdivided into a pair of copper atoms with six histidine ligands arranged trigonal prismatic. The pair probably represents the type-3 copper. The remaining copper has two histidine ligands. Its third site of co-ordination is formed by the pair of copper atoms. The fourth ligand may be OH- represented by a small protrusion of electron density. This copper probably is the type-2 copper. The symmetry of the trinuclear cluster is C2 and the ligands are supplied symmetrically by domains 1 and 3. However, domain 1 does not contain a type-1 copper and lacks the characteristic ligands. The unprecedented trinuclear cluster probably represents the oxygen binding and electron storage site.