The enzyme from Escherichia coli attacks a broad spectrum of phenolic compounds. The enzyme uses FADH2 as a substrate rather than a cofactor . FADH2 is provided by EC 1.5.1.36, flavin reductase (NADH) [5,6].
The enzyme from Escherichia coli attacks a broad spectrum of phenolic compounds. The enzyme uses FADH2 as a substrate rather than a cofactor [4]. FADH2 is provided by EC 1.5.1.36, flavin reductase (NADH) [5,6].
4-hydroxyphenylacetate, FAD, NADH, and O2 binding structures, overview, the binding and dissociation of flavin are accompanied by conformational changes of the loop between beta5 and beta6 and of the loop between beta8 and beta9, leading to preformation of part of the substrate-binding site involving Ser197 and Thr198. The latter loop further changes its conformation upon binding of 4-hydroxyphenylacetate and obstructs the active site from the bulk solvent. Arg100 is located adjacent to the putative oxygen-binding site and may be involved in the formation and stabilization of the C4a-hydroperoxyflavin intermediate
the oxygenase HpaB is a component of the 4-hydroxyphenylacetate 3-monooxygenase enzyme that is responsible for the hydroxylation of 4-hydroxyphenylacetate. It utilizes molecular oxygen and a reduced flavin, which is provided by HpaC, the second component of the enzyme
the oxygenase HpaB is a component of the 4-hydroxyphenylacetate 3-monooxygenase enzyme that is responsible for the hydroxylation of 4-hydroxyphenylacetate. It utilizes molecular oxygen and a reduced flavin, which is provided by HpaC, the second component of the enzyme
the hydroxylation of 4-hydroxyphenylacetate by this oxygenase is performed in two stages. Firstly, the reductase component of the enzyme, the small 16.1 kDa protein HpaC, reduces flavin with the use of NAD(P)H. Subsequently, reduced flavin is transported to the oxygenase component HpaB of 4HPA 3-monooxygenase by free diffusion where, together with the oxygen molecule, it is used for the oxygenation of substrates. HpaB determines the substrate specificity
the hydroxylation of 4-hydroxyphenylacetate by this oxygenase is performed in two stages. Firstly, the reductase component of the enzyme, the small 16.1 kDa protein HpaC, reduces flavin with the use of NAD(P)H. Subsequently, reduced flavin is transported to the oxygenase component HpaB of 4HPA 3-monooxygenase by free diffusion where, together with the oxygen molecule, it is used for the oxygenation of substrates. HpaB determines the substrate specificity
the oxygenase HpaB is a component of the 4-hydroxyphenylacetate 3-monooxygenase enzyme that is responsible for the hydroxylation of 4-hydroxyphenylacetate. It utilizes molecular oxygen and a reduced flavin, which is provided by HpaC, the second component of the enzyme
the oxygenase HpaB is a component of the 4-hydroxyphenylacetate 3-monooxygenase enzyme that is responsible for the hydroxylation of 4-hydroxyphenylacetate. It utilizes molecular oxygen and a reduced flavin, which is provided by HpaC, the second component of the enzyme
FAD is not tightly bound to HpaC, the flavin reductase component of the enzyme, and is bound in the groove in the extended and folded conformation, binding site structure, overview
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystal structures of ligand-free form, a binary complex with FAD, and a ternary complex with FAD and 4-hydroxyphenylacetate, to 2.0, 1.66 and 1.66 A resolution, respectively. Binding and dissociation of flavin are accompanied by conformational changes of the loop between beta5 and beta6 and of the loop between beta8 and beta9, leading to preformation of part of the substrate-binding site Ser197 and Thr198. The latter loop further changes its conformation upon binding of 4-hydroxyphenylacetate and obstructs the active site from the bulk solvent. Arg100 is located adjacent to the putative oxygen-binding site
sitting drop vapour diffusion method, 10 mg/ml protein in 0.01 M Tris-HCl, pH 7.6, equilibration against 0.5 ml of reservoir solution, different mixtures, overview, 20°C, well shaped single crystals, X-ray diffraction structure determination and analysis at 1.82 A resolution
flavin reductase component HpaC of 4-hydroxyphenylacetate 3-monooxygenase in a ternary complex with FAD and NAD+, mixing of 0.002-0.005 ml of a solution containing 25 mg/mL HpaCTt-FAD, 5 mM NADH, and 1 mM dithiothreitol with an equal volume of a reservoir solution containing 20% w/v PEG 1000, 10% w/v PEG 8000, and 10% v/v glycerol, and then equilibrating the solution against the reservoir solution, 2 days, X-ray diffraction structure determination and analysis at 1.65-3.3 A resolution
HpaB in three states: a ligand-free form, a binary complex with FAD, and a ternary complex with FAD and 4-hydroxyphenylacetate, mixing of 0.004 ml of protein solution containing 5 mg/ml protein and 1 mM DTT, with an equal volume of reservoir solution containing 1.5 M ammonium sulfate, 0.1 M Tris-HCl, pH 8.5, and 25% v/v glycerol, crystals appear within a few min and grow during 1-4 days to final size, complex crystal formation by soaking of crystals in 5 mM ligand containing solutions, X-ray diffraction structure determination and analysis at 1.66-2.07 A resolution
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
from membranes by solubilization with 0.1% Triton X-100, anion exchange chromatography, dialysis, exchange of detergent to dodecyl-beta-D-maltoside, another ion exchange chromatography, and gel filtration, further purification of HpaB by repetitive crystallization, method overview
Kim, S.H.; Hisano, T.; Iwasaki, W.; Ebihara, A.; Miki, K.
Crystal structure of the flavin reductase component (HpaC) of 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8: Structural basis for the flavin affinity