Correlating EPR and X-ray structural analysis of arsenite-inhibited forms of aldehyde oxidoreductase

J Biol Inorg Chem (2007) 12:353–366 DOI 10.1007/s00775-006-0191-9Two arsenite-inhibited forms of each of the aldehyde oxidoreductases from Desulfovibrio gigas and Desulfovibrio desulfuricans have been studied by X-ray crystallography and electron paramagnetic resonance (EPR) spectroscopy. The molybdenum site of these enzymes shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. Arsenite addition to active as-prepared enzyme or to a reduced desulfo form yields two different species called A and B, respectively, which show different Mo(V) EPR signals. Both EPR signals show strong hyperfine and quadrupolar couplings with an arsenic nucleus, which suggests that arsenic interacts with molybdenum through an equatorial ligand. X-ray data of single crystals prepared from EPR-active samples show in both inhibited forms that the arsenic atom interacts with the molybdenum ion through an oxygen atom at the catalytic labile site and that the sulfido ligand is no longer present. EPR and X-ray data indicate that the main difference between both species is an equatorial ligand to molybdenum which was determined to be an oxo ligand in species A and a hydroxyl/water ligand in species B. The conclusion that the sulfido ligand is not essential to determine the EPR properties in both Mo-As complexes is achieved through EPR measurements on a substantial number of randomly oriented chemically reduced crystals immediately followed by X-ray studies on one of those crystals. EPR saturation studies show that the electron transfer pathway, which is essential for catalysis, is not modified upon inhibition

X-ray crystal structure and EPR spectra of "arsenite-inhibited" Desulfovibriogigas aldehyde dehydrogenase: a member of the xanthine oxidase family

J. Am. Chem. Soc., 2004, 126 (28), pp 8614–8615 DOI: 10.1021/ja0490222X-ray crystallography has been used to determine the structure of arsenite-inhibited aldehyde dehydrogenase from Desulfovibrio gigas, a member of the xanthine oxidase family of mononuclear molybdenum enzymes. The structure shows an AsO3 moiety bound to the molybdenum atom of the active site through one of the oxygen atoms. A reduced sample of arsenite-inhibited aldehyde dehydrogenase has a Mo(V) signal that shows anisotropic hyperfine and quadrupole coupling to one arsenic atom. This signal has a strong resemblance with a previously reported signal for arsenite-inhibited xanthine oxidase

Purification and Preliminary Characterization of Tetraheme Cytochrome c3 and Adenylylsulfate Reductase from the Peptidolytic Sulfate-Reducing Bacterium Desulfovibrio aminophilus DSM 12254

Two proteins were purified and preliminarily characterized from the soluble extract of cells (310 g, wet weight) of the aminolytic and peptidolytic sulfate-reducing bacterium Desulfovibrio (D.) aminophilus DSM 12254. The iron-sulfur flavoenzyme adenylylsulfate (adenosine 5'-phosphosulfate, APS) reductase, a key enzyme in the microbial dissimilatory sulfate reduction, has been purified in three chromatographic steps (DEAE-Biogel A, Source 15, and Superdex 200 columns). It contains two different subunits with molecular masses of 75 and 18 kDa. The fraction after the last purification step had a purity index (A278nm / A388nm) of 5.34, which was used for further EPR spectroscopic studies. The D. aminophilus APS reductase is very similar to the homologous enzymes isolated from D. gigas and D. desulfuricans ATCC 27774. A tetraheme cytochrome c3 (His-heme iron-His) has been purified in three chromatographic steps (DEAE- Biogel A, Source 15, and Biogel-HTP columns) and preliminarily characterized. It has a purity index ([A553nm - A570nm]red / A280nm) of 2.9 and a molecular mass of around 15 kDa, and its spectroscopic characterization (NMR and EPR) has been carried out. This hemoprotein presents similarities with the tetraheme cytochrome c3 from Desulfomicrobium (Des.) norvegicum (NMR spectra, and N-terminal amino acid sequence)

Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo-C Interaction in a Biological System

J. Am. Chem. Soc., 2009, 131 (23), pp 7990–7998 DOI: 10.1021/ja809448rAldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a member of the xanthine oxidase(XO) family of mononuclear Mo-enzymes that catalyzes the oxidation of aldehydes to carboxylic acids. The molybdenum site in the enzymes of the XO family shows a distorted square pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. We report here steady-state kinetic studies of DgAOR with the inhibitors cyanide, ethylene glycol, glycerol, and arsenite, together with crystallographic and EPR studies of the enzyme after reaction with the two alcohols. In contrast to what has been observed in other members of the XO family, cyanide, ethylene glycol, and glycerol are reversible inhibitors of DgAOR. Kinetic data with both cyanide and samples prepared from single crystals confirm that DgAOR does not need a sulfido ligand for catalysis and confirm the absence of this ligand in the coordination sphere of the molybdenum atom in the active enzyme. Addition of ethylene glycol and glycerol to dithionite-reduced DgAOR yields rhombic Mo(V)EPR signals, suggesting that the nearly square pyramidal coordination of the active enzyme is distorted upon alcohol inhibition. This is in agreement with the X-ray structure of the ethylene glycol and glycerolinhibited enzyme, where the catalytically labile OH/OH2 ligand is lost and both alcohols coordinate the Mo site in a η2 fashion. The two adducts present a direct interaction between the molybdenum and one of the carbon atoms of the alcohol moiety, which constitutes the first structural evidence for such a bond in a biological system

Purification and Preliminary Characterization of Tetraheme Cytochrome and Adenylylsulf te Reductase from the Peptidolytic Sulfate-Reducing Bacterium Desulfovibrio aminophilus DSM 12254

Bioinorganic Chemistry and Applications Volume 3 (2005), Issue 1-2, Pages 81-91Two proteins were purified and preliminarily characterized from the soluble extract of cells (310 g, wet weight) of the aminolytic and peptidolytic sulfate-reducing bacterium Desulfovibrio (D.) aminophilus DSM 12254. The iron-sulfur flavoenzyme adenylylsulfate (adenosine 5"-phosphosulfate, APS)reductase, a key enzyme in the microbial dissimilatory sulfate reduction, has been purified in three chromatographic steps(DEAE-Biogel A, Source 15, and Superdex 200 columns). It contains two different subunits with molecular masses of 75 and 18 kDa. The fraction after the last purification step had a purity index (A278 ,m/A388 nm) of 5.34, which was used for further EPR spectroscopic studies. The D. aminophilus APS reductase is very similar to the homologous enzymes isolated from D. gigas and D. desulfuricans ATCC 27774. A tetraheme cytochrome c3 (His-heme iron-His) has been purified in three chromatographic steps (DEAE- Biogel A,Source 15, and Biogel-HTP columns) and preliminarily characterized. It has a purity index ([A.s.s3 nm" A570 nm]rcd/m280nm ox) of 2.9 and a molecular mass of around 15 kDa, and its spectroscopic characterization(NMR and EPR) has been carried out. This hemoprotein presents similarities with the tetraheme cytochrome c3 from Desulfomicrobium (Des.) norvegicurn (NMR spectra, and N-terminal amino acid sequence)

Representing Fitness Landscapes by Valued Constraints to Understand the Complexity of Local Search

Local search is widely used to solve combinatorial optimisation problems and to model biological evolution, but the performance of local search algorithms on different kinds of fitness landscapes is poorly understood. Here we introduce a natural approach to modelling fitness landscapes using valued constraints. This allows us to investigate minimal representations (normal forms) and to consider the effects of the structure of the constraint graph on the tractability of local search. First, we show that for fitness landscapes representable by binary Boolean valued constraints there is a minimal necessary constraint graph that can be easily computed. Second, we consider landscapes as equivalent if they allow the same (improving) local search moves; we show that a minimal normal form still exists, but is NP-hard to compute. Next we consider the complexity of local search on fitness landscapes modelled by valued constraints with restricted forms of constraint graph. In the binary Boolean case, we prove that a tree-structured constraint graph gives a tight quadratic bound on the number of improving moves made by any local search; hence, any landscape that can be represented by such a model will be tractable for local search. We build two families of examples to show that both the conditions in our tractability result are essential. With domain size three, even just a path of binary constraints can model a landscape with an exponentially long sequence of improving moves. With a treewidth two constraint graph, even with a maximum degree of three, binary Boolean constraints can model a landscape with an exponentially long sequence of improving moves

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

The power of Sherali-Adams relaxations for general-valued CSPs

We give a precise algebraic characterisation of the power of Sherali-Adams relaxations for solvability of valued constraint satisfaction problems to optimality. The condition is that of bounded width which has already been shown to capture the power of local consistency methods for decision CSPs and the power of semidefinite programming for robust approximation of CSPs. Our characterisation has several algorithmic and complexity consequences. On the algorithmic side, we show that several novel and many known valued constraint languages are tractable via the third level of the Sherali-Adams relaxation. For the known languages, this is a significantly simpler algorithm than the previously obtained ones. On the complexity side, we obtain a dichotomy theorem for valued constraint languages that can express an injective unary function. This implies a simple proof of the dichotomy theorem for conservative valued constraint languages established by Kolmogorov and Zivny [JACM'13], and also a dichotomy theorem for the exact solvability of Minimum-Solution problems. These are generalisations of Minimum-Ones problems to arbitrary finite domains. Our result improves on several previous classifications by Khanna et al. [SICOMP'00], Jonsson et al. [SICOMP'08], and Uppman [ICALP'13]

The power of Sherali-Adams relaxations for general-valued CSPs

We give a precise algebraic characterisation of the power of Sherali-Adams relaxations for solvability of valued constraint satisfaction problems to optimality. The condition is that of bounded width which has already been shown to capture the power of local consistency methods for decision CSPs and the power of semidefinite programming for robust approximation of CSPs. Our characterisation has several algorithmic and complexity consequences. On the algorithmic side, we show that several novel and many known valued constraint languages are tractable via the third level of the Sherali-Adams relaxation. For the known languages, this is a significantly simpler algorithm than the previously obtained ones. On the complexity side, we obtain a dichotomy theorem for valued constraint languages that can express an injective unary function. This implies a simple proof of the dichotomy theorem for conservative valued constraint languages established by Kolmogorov and Zivny [JACM'13], and also a dichotomy theorem for the exact solvability of Minimum-Solution problems. These are generalisations of Minimum-Ones problems to arbitrary finite domains. Our result improves on several previous classifications by Khanna et al. [SICOMP'00], Jonsson et al. [SICOMP'08], and Uppman [ICALP'13]