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1.14.14.9: 4-hydroxyphenylacetate 3-monooxygenase

This is an abbreviated version!
For detailed information about 4-hydroxyphenylacetate 3-monooxygenase, go to the full flat file.

Word Map on EC 1.14.14.9

Reaction

4-hydroxyphenylacetate
+
FADH2
 +
O2
=
3,4-Dihydroxyphenylacetate
 +
FAD
 +
H2O

Synonyms

4 HPA 3-hydroxyylase, 4-HPA hydroxylase, 4-hydroxyphenylacetate 3-hydroxylase, 4-hydroxyphenylacetic acid 3-hydroxylase, 4HPA 3-monooxygenase, 4HPA3H, C2-hpah, EC 1.14.13.3, HPA 3-hydroxylase, HpaB, hpaBC, HpaC, HPAH, More, p-hydroxyphenylacetate 3-hydroxylase, p-hydroxyphenylacetate hydroxylase, p-hydroxyphenylacetic 3-hydroxylase, TPY_2462, two-component p-hydroxyphenylacetate hydroxylase

ECTree

     1 Oxidoreductases
         1.14 Acting on paired donors, with incorporation or reduction of molecular oxygen
             1.14.14 With reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen into the other donor
                1.14.14.9 4-hydroxyphenylacetate 3-monooxygenase

Crystallization

Crystallization on EC 1.14.14.9 - 4-hydroxyphenylacetate 3-monooxygenase

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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
microbatch method, using 20% (W/v) PEG 400, 0.1 M sodium acetate pH 4.6 as a precipitant
-
models of the homotetrameric C2 component in complex with oxygen and presence or absence of 4-hydroxyphenylacetate. In random-acceleration molecular dynamics simulations, substrate 4-hydroxyphenylacetate can only reach the active center after the C4a-hydroperoxyflavin derivative of FMNH- is formed, requiring uptake of O2 at the active site before 4-hydroxyphenylacetate
in complex with FAD, to 1.94 A resolution
-
the active site is covered by a loop of great plasticity and strong tolerance towards extensive mutagenesis, but also is flexible loop and enables the entrance and stable binding of substrates into the active site
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
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
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