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PDBsum entry 2yyi
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Oxidoreductase
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PDB id
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2yyi
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References listed in PDB file
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Key reference
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Title
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Crystal structure of the oxygenase component (hpab) of the 4-Hydroxyphenylacetate 3-Monooxygenase from thermus thermophilus hb8.
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Authors
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S.H.Kim,
T.Hisano,
K.Takeda,
W.Iwasaki,
A.Ebihara,
K.Miki.
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Ref.
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J Biol Chem, 2007,
282,
33107-33117.
[DOI no: ]
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PubMed id
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Abstract
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The 4-hydroxyphenylacetate (4HPA) 3-monooxygenase is involved in the initial
step of the 4HPA degradation pathway and catalyzes 4HPA hydroxylation to
3,4-dihydroxyphenylacetate. This enzyme consists of two components, an oxygenase
(HpaB) and a reductase (HpaC). To understand the structural basis of the
catalytic mechanism of HpaB, crystal structures of HpaB from Thermus
thermophilus HB8 were determined in three states: a ligand-free form, a binary
complex with FAD, and a ternary complex with FAD and 4HPA. Structural analysis
revealed that 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 (Ser-197 and Thr-198). The latter loop further changes
its conformation upon binding of 4HPA and obstructs the active site from the
bulk solvent. Arg-100 is located adjacent to the putative oxygen-binding site
and may be involved in the formation and stabilization of the
C4a-hydroperoxyflavin intermediate.
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Figure 1.
FIGURE 1. A, crystal structure of HpaB homotetramer (the
unliganded form). Each monomer is shown in a different color. B,
a stereo view of the structure of HpaB monomer (the unliganded
form). The N-terminal domain (Ala-2—Leu-138), the middle
domain (Ala-139—Gly-266), the C-terminal domain
(Asn-267—Tyr-456), and the  -helical tail
(Asn-457—Ala-481) are shown in yellow, cyan, orange, and
magenta, respectively. The secondary structure elements are
labeled. All figures were generated using the PyMOL program.
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Figure 7.
FIGURE 7. A proposed catalytic mechanism of HpaB. (i) HpaB
binds reduced FAD (FADH[2]), which is supplied from HpaC. (ii)
FADH[2] reacts with an oxygen molecule and forms the
C4a-hydroperoxyflavin intermediate. (iii) Substrate binds to the
enzyme-C4a-hydroperoxyflavin complex. A hydroxyl group of the
C4a-hydroperoxyflavin intermediate is introduced into the ortho
position on the aromatic ring by electrophilic attack. (iv and
v) The dienone form of the product is rearomatized, and DHPA and
C4a-hydroxyflavin are formed. (vi) DHPA is released from the
enzyme. (vii) Water is eliminated from the C4a-hydroxyflavin.
(viii) Oxidized FAD is released from the enzyme and recycled to
HpaC to be reduced for the next catalytic cycle.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
282,
33107-33117)
copyright 2007.
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Secondary reference #1
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Title
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Crystallization and preliminary X-Ray analysis of the oxygenase component (hpab) of 4-Hydroxyphenylacetate 3-Monooxygenase from thermus thermophilus hb8.
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Authors
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S.H.Kim,
H.Miyatake,
T.Hisano,
W.Iwasaki,
A.Ebihara,
K.Miki.
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Ref.
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Acta Crystallogr Sect F Struct Biol Cryst Commun, 2007,
63,
556-559.
[DOI no: ]
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PubMed id
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Figure 1.
Reaction catalyzed by HpaB and HpaC. HpaC is an NADH:flavin
reductase that supplies reduced flavin to HpaB. Acta Crystallogr
Sect F Struct Biol Cryst Commun. 2007 July 1; 63(Pt 7):
556–559. Published online 2007 June 11. doi:
10.1107/S174430910702492X. Copyright [copyright] International
Union of Crystallography 2007
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Figure 2.
A crystal of HpaB obtained by the hanging-drop
vapour-diffusion method. The bar corresponds to 0.1 mm. Acta
Crystallogr Sect F Struct Biol Cryst Commun. 2007 July 1; 63(Pt
7): 556–559. Published online 2007 June 11. doi:
10.1107/S174430910702492X. Copyright [copyright] International
Union of Crystallography 2007
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The above figures are
reproduced from the cited reference
which is an Open Access publication published by the IUCr
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