Crystallization (Comment) | Organism |
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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 | Thermus thermophilus |
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 | Thermus thermophilus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
4-hydroxyphenylacetate + NAD(P)H + H+ + O2 | Thermus thermophilus | - |
3,4-dihydroxyphenylacetate + NAD(P)+ + H2O | - |
? | |
4-hydroxyphenylacetate + NAD(P)H + H+ + O2 | Thermus thermophilus HB8 / ATCC 27634 / DSM 579 | - |
3,4-dihydroxyphenylacetate + NAD(P)+ + H2O | - |
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Organism | UniProt | Comment | Textmining |
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Thermus thermophilus | - |
- |
- |
Thermus thermophilus | Q5SJP8 | - |
- |
Thermus thermophilus HB8 / ATCC 27634 / DSM 579 | - |
- |
- |
Thermus thermophilus HB8 / ATCC 27634 / DSM 579 | Q5SJP8 | - |
- |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
4-hydroxyphenylacetate + FADH2 + O2 = 3,4-dihydroxyphenylacetate + FAD + H2O | structural basis of the catalytic mechanism of HpaB | Thermus thermophilus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
4-hydroxyphenylacetate + NAD(P)H + H+ + O2 | - |
Thermus thermophilus | 3,4-dihydroxyphenylacetate + NAD(P)+ + H2O | - |
? | |
4-hydroxyphenylacetate + NAD(P)H + H+ + O2 | 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 | Thermus thermophilus | 3,4-dihydroxyphenylacetate + NAD(P)+ + H2O | - |
? | |
4-hydroxyphenylacetate + NAD(P)H + H+ + O2 | - |
Thermus thermophilus HB8 / ATCC 27634 / DSM 579 | 3,4-dihydroxyphenylacetate + NAD(P)+ + H2O | - |
? | |
4-hydroxyphenylacetate + NAD(P)H + H+ + O2 | 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 | Thermus thermophilus HB8 / ATCC 27634 / DSM 579 | 3,4-dihydroxyphenylacetate + NAD(P)+ + H2O | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | the enzyme consists of two components, an oxygenase HpaB and a reductase HpaC, structure analysis, overview | Thermus thermophilus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | binding structure, binding causes conformational changes, overview | Thermus thermophilus | |
NAD(P)H | - |
Thermus thermophilus |