Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Cholesterol oxidase (3 beta-hydroxysteroid oxidase, EC 1.1.3.6) is an FAD-dependent enzyme that carries out the oxidation and isomerization of steroids with a trans A : B ring junction. The crystal structure of the enzyme from Brevibacterium sterolicum has been determined using the method of isomorphous replacement and refined to 1.8 A resolution. The refined model includes 492 amino acid residues, the FAD prosthetic group and 453 solvent molecules. The crystallographic R-factor is 15.3% for all reflections between 10.0 A and 1.8 A resolution. The structure is made up of two domains: an FAD-binding domain and a steroid-binding domain. The FAD-binding domain consists of three non-continuous segments of sequence, including both the N terminus and the C terminus, and is made up of a six-stranded beta-sheet sandwiched between a four-stranded beta-sheet and three alpha-helices. The overall topology of this domain is very similar to other FAD-binding proteins. The steroid-binding domain consists of two non-continuous segments of sequence and contains a six-stranded antiparallel beta-sheet forming the "roof" of the active-site cavity. This large beta-sheet structure and the connections between the strands are topologically similar to the substrate-binding domain of the FAD-binding protein para-hydroxybenzoate hydroxylase. The active site lies at the interface of the two domains, in a large cavity filled with a well-ordered lattice of 13 solvent molecules. The flavin ring system of FAD lies on the "floor" of the cavity with N-5 of the ring system exposed. The ring system is twisted from a planar conformation by an angle of approximately 17 degrees, allowing hydrogen-bond interactions between the protein and the pyrimidine ring of FAD. The amino acid residues that line the active site are predominantly hydrophobic along the side of the cavity nearest the benzene ring of the flavin ring system, and are more hydrophilic on the opposite side near the pyrimidine ring. The cavity is buried inside the protein molecule, but three hydrophobic loops at the surface of the molecule show relatively high temperature factors, suggesting a flexible region that may form a possible path by which the substrate could enter the cavity. The active-site cavity contains one charged residue, Glu361, for which the side-chain electron density suggests a high degree of mobility for the side-chain. This residue is appropriately positioned to act as the proton acceptor in the proposed mechanism for the isomerization step.
J Mol Biol 1991 Jun 05
PMID:Crystal structure of cholesterol oxidase from Brevibacterium sterolicum refined at 1.8 A resolution. 205 87

Bone marrow stroma consists predominately of two cell types, macrophages and fibroblastoid stromal cells, which regulate the growth and differentiation of myelopoietic cells via the production of growth factors. We have previously shown that macrophages are more sensitive than fibroblastoid stromal cells (LTF cells) to the toxic effects of the benzene metabolite hydroquinone. In this study, the role of selective bioactivation and/or deactivation in the macrophage-selective effects of hydroquinone was examined. LTF and macrophage cultures were incubated with 10 microM [14C]hydroquinone to examine differential bioactivation. After 24 hr, the amount of 14C covalently bound to acid-insoluble macromolecules was determined. Macrophages had 16-fold higher levels of macromolecule-associated 14C than did LTF cells. Additional experiments revealed that hydroquinone bioactivation to covalent-binding species was hydrogen peroxide dependent in macrophage homogenates. Covalent binding in companion LTF homogenates was minimal, even in the presence of excess hydrogen peroxide. These data suggest that a peroxidative event was responsible for bioactivation in macrophages and, in agreement with this, macrophages contained detectable peroxidase activity whereas LTF cells did not. Bioactivation of [14C]hydroquinone to protein-binding species by peroxidase was confirmed utilizing purified human myeloperoxidase in the presence of hydrogen peroxide and ovalbumin as a protein source. High performance liquid chromatographic analysis of incubations containing purified myeloperoxidase, hydroquinone, and hydrogen peroxide showed that greater than 90% of hydroquinone was removed and could be detected stoichometrically as 1,4-benzoquinone. 1,4-Benzoquinone was confirmed as a reactive metabolite formed from hydroquinone in macrophage incubations using excess GSH and trapping the reactive quinone as its GSH conjugate, which was measured by high performance liquid chromatography with electrochemical detection. The activity of DT-diaphorase, a quinone reductase that has been invoked as a protective mechanism in quinone-induced toxicity, was 4-fold higher in LTF cells than macrophages. These data suggest that the macrophage-selective toxicity of hydroquinone results from higher levels of peroxidase-mediated bioactivation and/or lower levels of DT-diaphorase-mediated detoxification.
Mol Pharmacol 1990 Feb
PMID:Bone marrow stromal cell bioactivation and detoxification of the benzene metabolite hydroquinone: comparison of macrophages and fibroblastoid cells. 215 73

The structural requirements for stimulation of adenosine A1 agonist binding by 2-amino-3-benzoylthiophenes and related compounds were investigated. Slowing of the dissociation of [3H]N6cyclohexyladenosine binding was used as a specific measure of the allosteric effects of these compounds. The thiophene ring could be replaced with benzene but not with several nitrogen-containing heterocycles. The 2-amino group was required, and at least one hydrogen on the amino group appeared to be necessary for activity. The keto carbonyl was also essential. Alkyl substitution at the 4-position of the thiophene ring increased activity, whereas 5-position substitution appeared to have little effect. Activity was also increased by various substitutions on the phenyl ring, with 3-(trifluoromethyl) showing optimal activity. The phenyl ring could be replaced with cyclohexyl without major loss of activity. 1-Aminofluoren-9-one, a conformationally locked derivative, was active. Based in part in the latter observation, the active conformation is proposed to have an intramolecular hydrogen bond between the amino nitrogen and the carbonyl oxygen. Because the 2-amino-3-benzoylthiophenes showed competitive adenosine antagonism as well as allosteric enhancement, their affinities as competitive inhibitors of [3H]8-cyclopentyl-1,3-dipropylxanthine binding to A1 receptors were also assessed. Structure-activity relationships for competitive antagonism were distinct from those for allosteric enhancement, with ratios between the two activities varying by more than 1000-fold. Of the analogs tested, (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)phenyl]methanone (PD 81,723) had the most favorable ratio of enhancement to antagonism.
Mol Pharmacol 1990 Dec
PMID:Structure-activity relationships for enhancement of adenosine A1 receptor binding by 2-amino-3-benzoylthiophenes. 225 Jun 67

Non-bonded potentials for the aryl..aryl interaction have been derived using crystal structure data of a number of small aromatic molecules. The potentials, based on atom-centered interactions, give an accurate reproduction of the benzene crystal geometry and sublimation energy when used in conjunction with coulombic energies evaluated using point atomic charges. An examination of the charge distribution on benzene suggested values of 0.13e (H) and -0.13e (C) to be suitable. The transferability of the potentials has been shown by prediction of crystal geometries and sublimation energies of other hydrocarbon molecules and, with additional interactions for the oxygen atom included, preliminary polymer crystal structure calculations have been carried out. These demonstrate the validity of the derived parameters by successfully predicting crystallographic unit cell dimensions and ring conformations in the poly(phenylene oxide) and poly(aryl ether ketone) crystals.
J Comput Aided Mol Des 1990 Sep
PMID:Molecular modelling of poly(aryl ether ketones). I. Aryl..aryl interactions in crystal structures. 228 Feb 65

The arene oxides formation energy for about 30 benzene derivatives were calculated in frames of MO LCAO method. The benzene derivatives are typical substrates of cytochrome P-450 catalyzed aromatic oxidation. The relationship between the formation energy of tetrahedral type arene oxides and the relative reactivity of substrates toward microsomal hydroxylation was found. The formation energy of arene oxides correlates also with toxicity of benzene derivatives. Some limitations of the oxenoid model are discussed.
Mol Biol (Mosk)
PMID:[The oxenoid model of the mechanism of activating molecular oxygen by cytochrome p450: the role of substrate structure]. 229 Apr 28

Using synchrotron radiation, the X-ray diffraction intensities of crystals of p-hydroxy-benzoate hydroxylase, complexed with the substrate p-hydroxybenzoate, were measured to a resolution of 1.9 A. Restrained least-squares refinement alternated with rebuilding in electron density maps yielded an atom model of the enzyme-substrate complex with a crystallographic R-factor of 15.6% for 31,148 reflections between 6.0 and 1.9 A. A total of 330 solvent molecules was located. In the final model, only three residues have deviating phi-psi angle combinations. One of them, the active site residue Arg44, has a well-defined electron density and may be strained to adopt this conformation for efficient catalysis. The mode of binding of FAD is distinctly different for the different components of the coenzyme. The adenine ring is engaged in three water-mediated hydrogen bonds with the protein, while making only one direct hydrogen bond with the enzyme. The pyrophosphate moiety makes five water-mediated versus three direct hydrogen bonds. The ribityl and ribose moieties make only direct hydrogen bonds, in all cases, except one, with side-chain atoms. The isoalloxazine ring also makes only direct hydrogen bonds, but virtually only with main-chain atoms. The conformation of FAD in p-hydroxybenzoate hydroxylase is strikingly similar to that in glutathione reductase, while the riboflavin-binding parts of these two enzymes have no structural similarity at all. The refined 1.9 A structure of the p-hydroxybenzoate hydroxylase-substrate complex was the basis of further refinement of the 2.3 A structure of the enzyme-product complex. The result was a final R-factor of 16.7% for 14,339 reflections between 6.0 and 2.3 A and an improved geometry. Comparison between the complexes indicated only small differences in the active site region, where the product molecule is rotated by 14 degrees compared with the substrate in the enzyme-substrate complex. During the refinements of the enzyme-substrate and enzyme-product complexes, the flavin ring was allowed to bend or twist by imposing planarity restraints on the benzene and pyrimidine ring, but not on the flavin ring as a whole. The observed angle between the benzene ring and the pyrimidine ring was 10 degrees for the enzyme-substrate complex and 19 degrees for the enzyme-product complex. Because of the high temperature factors of the flavin ring in the enzyme-product complex, the latter value should be treated with caution. Six out of eight peptide residues near the flavin ring are oriented with their nitrogen atom pointing towards the ring.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1989 Aug 20
PMID:Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 A resolution. Analysis of the enzyme-substrate and enzyme-product complexes. 255 83

The backbone conformations of the cyclic moieties of 1-[beta-mercaptopropionic acid]-oxytocin [( Mpa1]-OT), [1-beta-mercaptopropionic acid]-arginine-vasopressin [( Mpa1]-AVP), [1-(beta'-mercapto-beta,beta-cyclopentamethylene)propionic acid]-arginine-vasopressin [( Cpp1]-AVP), and [1-thiosalicylic acid]-arginine-vasopressin [( Ths1]-AVP) have been analyzed by means of molecular mechanics. In these calculations, the side chains were simulated by pseudoatoms. For the three last compounds, the calculations were also performed on the whole molecules, in order to shed light on the differences in their biological activity. Their starting conformations were obtained by attaching the acyclic tail and side chains to the lowest energy conformations of the cyclic parts. In the case of [Ths1]-AVP, however, other starting conformations were also examined, which were obtained by attaching the planar benzene ring to the lowest energy conformations of [Mpa1]-AVP. In the calculations, all the degrees of freedom were relaxed and Weiner's force field was used, the parameters required for the benzene parts of [Ths1]-AVP being determined from the experimental data available, as well as from the results of molecular dynamics calculations on the model compounds. The lowest energy conformations of [Mpa1]-AVP and [Cpp1]-AVP are similar, while [Ths1]-AVP differs from them near the disulphide region, due to the presence of a planar benzene ring. Interactions involving the charged guanidine group of arginine make, in each case, an important contribution to the conformational energy. A model description of the shapes of the oxytocin and vasopressin ring has been proposed, which is based on the cyclohexane geometry. This description is in good correlation with the energetics of the conformations corresponding to different shapes.
J Comput Aided Mol Des 1989 Jan
PMID:Molecular mechanics calculations on deaminooxytocin and on deamino-arginine-vasopressin and its analogues. 271 90

The X-ray structures of two new 2,6-disubstituted benzamides, i.e., remoxipride hydrochloride monohydrate [-)-(S)-3-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2,6-dimethoxybenza mide hydrochloride monohydrate) and FLA 797 [-)-(S)-3-bromo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-6-methoxysalicylamide ), have been determined as well as the distribution coefficients. The difference in dopamine receptor blocking activity is discussed in terms of lipophilicity and solid state conformations of the two benzamides. The major difference between the solid state conformations lies in the orientation of the carboxamide moiety. In remoxipride the carbonyl group is oriented almost perpendicularly to the benzene ring, thus preventing the formation of a hydrogen-bonded pseudo-ring between the amide hydrogen and the methoxy group found in other types of o-methoxybenzamides. In FLA 797, however, this pseudo-ring is present in the planar conformation of the salicylamide moiety. This conformation is further stabilized by a hydrogen bond between the phenol group and the carbonyl oxygen. The side chain in remoxipride adopts an extended conformation in contrast to FLA 797, where the side chain has a folded conformation. The crystal structures are related to current topographic dopamine receptor models developed from more rigid antidopaminergic compounds. Based on these comparisons, it is suggested that benzamides having an N-ethyl-2-pyrrolidinylmethyl side chain interact with the receptor in the folded conformation. The binding affinity is thought to be further increased by the planar conformation of the salicylamide moiety present in FLA 797, which permits an efficient pi-pi stacking interaction.
Mol Pharmacol 1986 Oct
PMID:Solid state conformations and antidopaminergic effects of remoxipride hydrochloride and a closely related salicylamide, FLA 797, in relation to dopamine receptor models. 294 89

The oxidation of phenol catalyzed by human myeloperoxidase and horseradish peroxidase resulted in extensive binding of phenol-derived metabolites to boiled rat liver protein. This binding paralleled closely the removal of phenol from the incubations and was inhibited from 83 to 99% by the addition of the antioxidants, ascorbate and glutathione, suggesting that metabolism and binding were occurring via a one-electron oxidation pathway. Metabolic studies employing both human myeloperoxidase and horseradish peroxidase resulted in the identification of 4,4'-biphenol and diphenoquinone as the principal identifiable metabolites. The addition of reduced glutathione to incubations containing horseradish peroxidase resulted in the formation of two conjugate species. These conjugate species were identified by fast atom bombardment mass spectrometry to be glutathione conjugates of diphenoquinone. The major gluthathione conjugate was identified as 3-(glutathion-S-yl)-4,4'-biphenol by NMR spectroscopy. These results suggest that the formation of highly reactive species through the peroxidase-mediated metabolism of phenol and other phenolic compounds could play an important role in the hematopoietic toxicity observed during chronic benzene exposure.
Mol Pharmacol 1986 Dec
PMID:Metabolic activation of phenol by human myeloperoxidase and horseradish peroxidase. 302 15

Various hydrocarbons were synthesized by high-frequency discharge in a primordial terrestrial model atmosphere. The products were extracted by benzene or methanol and analyzed by GC-MS. The mean carbon chain length of the hydrocarbons formed by the discharge through pure CH4 gas was less than 6. Benzene was also obtained. Some isomers were obtained for each of the hydrocarbons containing a given number of carbons. When a small amount of C2H2 was added to the CH4, longer chain compounds were formed, as compared with discharge in CH4 only. However, when the amount of C2H2 was increased, unextractable high molecular weight compounds were produced. The amounts of products decreased as the mixing ratio of CO2 to CH4 increased. No hydrocarbons were detected when the ratio of CO2/CH4 exceeded 1.
J Mol Evol 1986
PMID:Synthesis of hydrocarbons under presumed prebiotic conditions using high-frequency discharge. 309 42


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