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Structural models determined by X-ray crystallography play a central role in understanding the catalytic mechanism of enzymes. However, X-ray radiation generates hydrated electrons that can cause significant damage to the active sites of metalloenzymes. In the present study, crystal structures of the multicopper oxidases (MCOs) CueO from Escherichia coli and laccase from a metagenome were determined. Diffraction data were obtained from a single crystal under low to high X-ray dose conditions. At low levels of X-ray exposure, unambiguous electron density for an O atom was observed inside the trinuclear copper centre (TNC) in both MCOs. The gradual reduction of copper by hydrated electrons monitored by measurement of the Cu K-edge X-ray absorption spectra led to the disappearance of the electron density for the O atom. In addition, the size of the copper triangle was enlarged by a two-step shift in the location of the type III coppers owing to reduction. Further, binding of O2 to the TNC after its full reduction was observed in the case of the laccase. Based on these novel structural findings, the diverse resting structures of the MCOs and their four-electron O2-reduction process are discussed.

Aim: To assess the glucose-lowering effects of monotherapy with the glucokinase activator AZD1656 in Japanese patients with type 2 diabetes mellitus., Methods: This was a randomized, double-blind, placebo-controlled study performed in Japan (NCT01152385). Patients (n = 224) were randomized to AZD1656 (40-200, 20-140 or 10-80 mg titrated doses) or placebo. The primary variable was the placebo-corrected change from baseline to 4 months in glycated haemoglobin (HbA1c). Effects on fasting plasma glucose (FPG) and safety were also assessed., Results: HbA1c was reduced numerically from baseline by 0.3-0.8% with AZD1656 and by 0.1% with placebo over the first 2 months of treatment, after which effects of AZD1656 started to decline. The changes from baseline to 4 months in HbA1c were not significant for the AZD1656 40-200 mg group versus placebo [mean (95% CI) placebo-corrected change: -0.22 (-0.65, 0.20)%; p = 0.30]. Formal significance testing was not carried out for the other two AZD1656 dose groups. A higher percentage of patients on AZD1656 achieved HbA1c ≤ 7% after 4 months versus placebo, but responder rates were low. Results for FPG reflected those for HbA1c. Cases of hypoglycaemia were rare with AZD1656 (one patient) and no safety concerns were raised., Conclusions: Although initially favourable plasma glucose reductions were observed, there was a loss of effect over time with sustained AZD1656 treatment. The study design did not allow an evaluation of the reasons for this lack of long-term efficacy., (© 2013 John Wiley & Sons Ltd.)

The core domain of a human histidine decarboxylase mutant was purified and cocrystallized with the inhibitor L-histidine methyl ester. Using synchrotron radiation, a data set was collected from a single crystal at 100 K to 1.8 Å resolution. The crystal belonged to space group C2, with unit-cell parameters a = 215.16, b = 112.72, c = 171.39 Å, β = 110.3°. Molecular replacement was carried out using the structure of aromatic L-amino-acid decarboxylase as a search model. The crystal contained three dimers per asymmetric unit, with a Matthews coefficient (V(M)) of 3.01 Å(3) Da(-1) and an estimated solvent content of 59.1%.

Coiled-coil DIX1 (Ccd1) is a positive regulator that activates the canonical Wnt signalling pathway by inhibiting the degradation of the key signal transducer β-catenin. The C-terminal DIX domain of Ccd1 plays an important role in the regulation of signal transduction through homo-oligomerization and protein complex formation with other DIX domain-containing proteins, i.e. axin and dishevelled proteins. Here, the expression, purification, crystallization and X-ray data collection of the Ccd1 DIX domain are reported. The crystals of the Ccd1 DIX domain belonged to space group P2(1)2(1)2(1), with unit-cell parameters a=72.9, b=75.7, c=125.6 Å. An X-ray diffraction data set was collected at 3.0 Å resolution.

Cytochrome c (cyt c) is an electron-transfer protein in the respiratory chain of mitochondria. It is known to form polymers, but its polymerization mechanism is still unknown. Dimeric and trimeric cyt c from horse were successfully crystallized by the sitting-drop vapour-diffusion method using polyethylene glycol as a precipitating reagent. The crystal of dimeric cyt c belonged to space group P1, with unit-cell parameters a = 41.8, b = 56.3, c = 60.8 Å, α = 66.3, β = 89.9, γ = 73.7°, whereas that of trimeric cyt c belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 57.2, b = 95.7, c = 130.9 Å. Initial structure models showed that the crystals of dimeric and trimeric cyt c contained two dimers and two trimers, respectively, in the asymmetric unit.

Ethanolamine ammonia-lyase (EAL) catalyzes the adenosylcobalamin-dependent conversion of ethanolamine to acetaldehyde and ammonia. The wild-type enzyme shows a very low solubility. N-terminal truncation of the Escherichia coli EAL beta-subunit dramatically increases the solubility of the enzyme without altering its catalytic properties. Two deletion mutants of the enzyme [EAL(betaDelta4-30) and EAL(betaDelta4-43)] have been overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method. Crystals of EAL(betaDelta4-30) and EAL(betaDelta4-43) diffracted to approximately 8.0 and 2.1 A resolution, respectively.

Ferredoxin-NADP(+) oxidoreductase encoded by Bacillus subtilis yumC has been purified and successfully crystallized in complex with NADP(+) in two forms. Diffraction data from crystals of these two forms were collected at resolutions of 1.8 and 1.9 A. The former belonged to space group P2(1)2(1)2, with unit-cell parameters a = 63.90, b = 135.72, c = 39.19 A, and the latter to space group C2, with unit-cell parameters a = 207.47, b = 64.85, c = 61.12 A, beta = 105.82 degrees. The initial structure was determined by the molecular-replacement method using a thioredoxin reductase-like protein as a search model.

A putative two-domain-type laccase retrieved from a metagenome was successfully crystallized using the sitting-drop vapour-diffusion method. Data were collected to a resolution of 1.7 A at 100 K using synchrotron radiation. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 74.67, b = 100.95, c = 124.11 A. The self-rotation function showed the presence of a noncrystallographic threefold axis in the structure. The presence of one trimer in the asymmetric unit yielded a Matthews coefficient (V(M)) of 2.05 A(3) Da(-1) and a solvent content of 40%.

Class II release factor 3 (RF3) from the sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki F, which promotes rapid dissociation of a class I release factor, has been overexpressed, purified and crystallized in complex with GDP at 293 K using the sitting-drop vapour-diffusion method. A data set was collected to 1.8 A resolution from a single crystal at 100 K using synchrotron radiation. The crystal belongs to space group P1, with unit-cell parameters a = 47.39, b = 82.80, c = 148.29 A, alpha = 104.21, beta = 89.78, gamma = 89.63 degrees . The asymmetric unit contains four molecules of the RF3-GDP complex. The Matthews coefficient was calculated to be 2.3 A(3) Da(-1) and the solvent content was estimated to be 46.6%.

Axin is a negative regulator of the canonical Wnt signalling pathway that mediates the phosphorylation of beta-catenin by glycogen synthase kinase 3beta. The DIX domain of rat axin, which is important for its homooligomerization and interactions with other regulators in the Wnt pathway, was purified and crystallized by the sitting-drop vapour-diffusion technique using polyethylene glycol 6000 and lithium sulfate as crystallization agents. Crystals belong to space group P6(1) or P6(5), with unit-cell parameters a = b = 91.49, c = 84.92 A. An X-ray diffraction data set has been collected to a nominal resolution of 2.9 A.

Objective: Crescent formation in the renal glomerulus is a typical manifestation of progressive glomerulopathy associated with fatal renal failure; therefore, its prevention is of clinical importance. Little is known about the pathogenic mechanism for crescent formation. This study was undertaken in an attempt to identify the events that are critical for crescent formation in immune complex crescentic glomerulonephritis (CGN) by analyzing a novel mutant strain of mice., Methods: A spontaneous mutant strain of mice was isolated from the autoimmune-prone strain EOD, which stably develops fatal CGN. The mutant phenotypes were assessed histopathologically, hematologically, and immunologically. The mutation was searched for with positional cloning using microsatellite markers., Results: Compared with wild-type EOD (WT-EOD) mice, mutant EOD (mut-EOD) mice showed marked improvement in CGN in conjunction with an improvement in spontaneous mortality. In WT-EOD mice, an inverse correlation between blood urea nitrogen concentration and blood platelet count and massive accumulation of platelets in the glomerulus were evident, suggesting that an accumulation of platelets in the glomerulus contributes to the progression of CGN. The mutant platelets showed an abnormal aggregation in response to collagen and thrombin, associated with a bleeding tendency in mut-EOD mice. Genetic analysis revealed a deleterious mutation in the cappuccino gene (cno), which encodes a protein that belongs to a complex called the biogenesis of lysosome-related organelle complex 1 and is profoundly involved in platelet function. Morphologic examination revealed a partial defect in dense body formation in the delta-granule of platelets., Conclusion: The present findings suggest that platelet functions have a critical role in crescent formation in autoimmune GN.

CooA, a homodimeric haem-containing protein, is responsible for transcriptional regulation in response to carbon monoxide (CO). It has a b-type haem as a CO sensor. Upon binding CO to the haem, CooA binds promoter DNA and activates expression of genes for CO metabolism. CooA from Carboxydothermus hydrogenoformans has been overexpressed in Escherichia coli, purified and crystallized by the vapour-diffusion method. The crystal belongs to space group P2(1), with unit-cell parameters a = 61.8, b = 94.7, c = 92.8 angstroms, beta = 104.8 degrees. The native and anomalous difference Patterson maps indicated that two CooA dimers are contained in the asymmetric unit and are related by a translational symmetry almost parallel to the c axis.

The crystal structure of a conserved hypothetical protein, TTHA0849 from Thermus thermophilus HB8, has been determined at 2.4 A resolution as a part of a structural and functional genomics project on T. thermophilus HB8. The main-chain folding shows a compact alpha+beta motif, forming a hydrophobic cavity in the molecule. A structural similarity search reveals that it resembles those steroidogenic acute regulatory proteins that contain the lipid-transfer (START) domain, even though TTHA0849 shows comparatively weak sequence identity to polyketide cyclases. However, the size of the ligand-binding cavity is distinctly smaller than other START domain-containing proteins, suggesting that it catalyses the transfer of smaller ligand molecules.

DNA photolyase is a unique flavoenzyme that repairs UV-induced DNA lesions using the energy of visible light. Anacystis nidulans photolyase contains a light-harvesting chromophore, 8-hydroxy-5-deazaflavin (8-HDF), and flavin adenine dinucleotide (FAD) which, in contrast to the 8-HDF chromophore, is indispensable for catalytic activity. This work reports the crystallization and structure at 1.8 A resolution of DNA photolyase devoid of its 8-HDF chromophore (apophotolyase). The overall three-dimensional structure is similar to that of the holoenzyme, indicating that the presence of 8-HDF is not essential for the correct folding of the enzyme. Structural changes include an additional phosphate group, a different conformation for Arg11 and slight rearrangements of Met47, Asp101 and Asp382, which replace part of the 8-HDF molecule in the chromophore-binding pocket. The apophotolyase can be efficiently reconstituted with synthetic 8-hydroxy-5-deazariboflavin, despite the orientation of Arg11 and the presence of the phosphate group in the 8-HDF pocket. Red light or X-rays reduced the FAD chromophore in apophotolyase crystals, as observed by single-crystal spectrophotometry. The structural effects of FAD reduction were determined by comparison of three data sets that were successively collected at 100 K, while the degree of reduction was monitored online by changes in the light absorption of the crystals. X-ray-induced conformational changes were confined to the active site of the protein. They include sub-ångström movements of the O(2) and N(5) atoms of the flavin group as well as the O(delta) atoms of the surrounding amino acids Asp380 and Asn386.

We identified an age effect on biopterin and biogenic amine metabolites in the cerebrospinal fluid from 56 neurologically normal patients, aged 1 mo to 80 y. The levels of total and reduced forms of biopterin, homovanillic acid, and 5-hydroxyindoleacetic acid were found to peak during the first year of life. The levels of these metabolites then gradually decreased, plateauing at the age of 20 y. We found significant correlations between biopterin, homovanillic acid, and 5-hydroxy-indoleacetic acid.