the cleavage mechanism of the aliphatic C-C bond catalyzed by 2,4'-dihydroxyacetophenone dioxygenase, quantum mechanics/molecular mechanics calculations. The reactant complex may firstly undergo a triplet-quintet crossing to initiate the reaction and then the subsequent chemistry mainly occurs on the quintet state surface, modeling of the enzyme-substrate complex, overview
enzyme DAD is very unusual in that it is involved in C-C bond cleavage in a substituent of the aromatic ring instead of cleaving within the aromatic ring of the substrate
enzyme DAD is very unusual in that it is involved in C-C bond cleavage in a substituent of the aromatic ring instead of cleaving within the aromatic ring of the substrate
enzyme DAD is very unusual in that it is involved in C-C bond cleavage in a substituent of the aromatic ring instead of cleaving within the aromatic ring of the substrate
enzyme DAD is very unusual in that it is involved in C-C bond cleavage in a substituent of the aromatic ring instead of cleaving within the aromatic ring of the substrate
enzyme DAD is very unusual in that it is involved in C-C bond cleavage in a substituent of the aromatic ring instead of cleaving within the aromatic ring of the substrate
enzyme DAD is very unusual in that it is involved in C-C bond cleavage in a substituent of the aromatic ring instead of cleaving within the aromatic ring of the substrate
enzyme DAD is very unusual in that it is involved in C-C bond cleavage in a substituent of the aromatic ring instead of cleaving within the aromatic ring of the substrate
enzyme DAD is very unusual in that it is involved in C-C bond cleavage in a substituent of the aromatic ring instead of cleaving within the aromatic ring of the substrate
one non-heme iron per enzyme subunit. The catalytic iron is coordinated by three histidine residues, H76, H78 and H114 in strands II and VII, within a buried active-site cavity, and an additional ligand
the enzyme contains 1.69 mol of non-heme iron per mol of enzyme, His77, His79, His115, and Glu96 in the cupin fold are putative metal ligands, Fe2+ weakly activates the enzyme
anaerobic reconstitution of the purified enzyme with five equivalents of Fe2+ results in an increase in activity of 7% which increases to 13% upon overnight incubation in air
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging drop vapour diffusion method, mixing of 5 mg/ml protein in 50 mM Tris, pH 7.3, 100 mM NaCl, and mM 4-hydroxybenzoic acid or 0.1 mM 4-aminobenzamidine, with reservoir solution containing 0.1 M sodium cacodylate pH 5.9-6.8 and 1.1-1.6 M sodium acetate, method optimization, X-ray diffraction structure determination and analysis at 1.88 A resolution. The use of limited chymotrypsinolysis, which apparently results in removal of the first 20 or so N-terminal residues of DAD, is necessary for crystallization of the protein
purified recombinant detagged chymotrypsinolysed enzyme, 5 mg/ml protein in 50 mM Tris, pH 7.5, 100 mM NaCl, 1 mM 2-mercaptoethanol is mixed with a solution containing 10% w/v PEG 1000 and 10% PEG 8000, X-ray diffraction structure determination and analysis at 2.2 A resolution. The use of limited chymotrypsinolysis, which apparently results in removal of the first 20 or so N-terminal residues of DAD, is necessary for crystallization of the protein
purified recombinant His-tagged chymotrypsinolysed enzyme, 5 mg/ml protein in 50 mM Tris, pH 7.5, 100 mM NaCl, 1 mM 2-mercaptoethanol is mixed with a solution containing 10% w/v PEG 1000 and 10% PEG 8000, X-ray diffraction structure determination and analysis at 2.2 A resolution. The use of limited chymotrypsinolysis, which apparently results in removal of the first 20 or so N-terminal residues of DAD, is necessary for crystallization of the protein
recombinant enzyme 39fold from Escherichia coli strain JM109 by heat treatment, ammonium sulfate fractionation, gel filtration, and anion exchange chromatography, native enzyme 17fold from strain AZ11 by gel filtration, anion exchange and hydrophobic interaction chromatography
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene cloned, sequenced and expressed in Escherichia coli N-4830, nucleotide sequence of the gene dad deposited in the GenBank (r)/EMBL/DDBJ nucleotide sequence databases
Unraveling the crucial role of single active water molecule in the oxidative cleavage of aliphatic C-C bond of 2,4?-dihydroxyacetophenone catalyzed by 2,4'-dihydroxyacetophenone dioxygenase enzyme A quantum mechanics/molecular mechanics investigation