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References listed in PDB file
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Key reference
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Title
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Lys42 and ser42 variants of p-Hydroxybenzoate hydroxylase from pseudomonas fluorescens reveal that arg42 is essential for NADPH binding.
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Authors
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M.H.Eppink,
H.A.Schreuder,
W.J.Van berkel.
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Ref.
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Eur J Biochem, 1998,
253,
194-201.
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PubMed id
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Abstract
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The conserved Arg42 of the flavoprotein p-hydroxybenzoate hydroxylase is located
at the entrance of the active site in a loop between helix H2 and sheet E1 of
the FAD-binding domain. Replacement of Arg42 by Lys or Ser decreases the
turnover rate of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens by
more than two orders of magnitude. Rapid reaction kinetics show that the low
activity of the Arg42 variants results from impaired binding of NADPH. In
contrast to an earlier conclusion drawn for p-hydroxybenzoate hydroxylase from
Acinetobacter calcoaceticus, substitution of Arg42 with Ser42 in the enzyme from
P. fluorescens hardly disturbs the binding of FAD. Crystals of
[Lys42]p-hydroxybenzoate hydroxylase complexed with 4-hydroxybenzoate diffract
to 0.22-nm resolution. The structure of the Lys42 variant is virtually
indistinguishable from the native enzyme with the flavin ring occupying the
interior position within the active site. Lys42 in the mutant structure
interacts indirectly via a solvent molecule with the 3-OH of the adenosine
ribose moiety of FAD. Substrate perturbation difference spectra suggest that the
Arg42 replacements influence the solvent accessibility of the flavin ring in the
oxidized enzyme. In spite of this, the Arg42 variants fully couple enzyme
reduction to substrate hydroxylation. Sequence-comparison studies suggest that
Arg42 is involved in binding of the 2'-phosphoadenosine moiety of NADPH.
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Secondary reference #1
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Title
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Crystal structure of p-Hydroxybenzoate hydroxylase reconstituted with the modified FAD present in alcohol oxidase from methylotrophic yeasts: evidence for an arabinoflavin.
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Authors
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W.J.Van berkel,
M.H.Eppink,
H.A.Schreuder.
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Ref.
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Protein Sci, 1994,
3,
2245-2253.
[DOI no: ]
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PubMed id
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Figure 3.
Fig. 3. Superposition of thestructurs of
hea-FAD-containingenzyme-4hydroxy-
benzoatecomplexandthenative enzyme-
2,4-dihydroxybenzoatecomplex.The
structure of the4-hydroxybenzoatecom-
plexwitha-FADisdrawnwithsolid
bonds;thestructure of the 2,4-dihydroxy-
benzoatecomplexwithnaturalFAD s
drawnwithopenbonds.The view is from
theflavinringtowardtheribitylchain.
Brokenlinesindicatethehydrogenbond
between the02' hydroxyl groupandthe
OEl of Gln 102, is presentinboth
complexes.
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Figure 5.
Fig. 5. Stereodiagram of hesuperosi-
ion of native2,4-dihydroxybenzoate
complexedp-hydroxybenzoate hydroxylase
Schreuderetal., 1994; graybonds)and
hea-FAD-containingenzyme-substrate
complex(blackbonds).Theflavinring
ccupiesthe``out''postioninbothcom-
lexes, buttheflavinring is slightly fur-
heroutinthe2,4-dihydroxybenzoate
complex,presumably because of ahydro-
genbondcontact,indicatedbyabroken
line,between the 2-hydroxygroup of he
substrateanalogandthe N3 of thelavin
ring.
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The above figures are
reproduced from the cited reference
which is an Open Access publication published by the Protein Society
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Secondary reference #2
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Title
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Crystal structures of wild-Type p-Hydroxybenzoate hydroxylase complexed with 4-Aminobenzoate,2,4-Dihydroxybenzoate, And 2-Hydroxy-4-Aminobenzoate and of the tyr222ala mutant complexed with 2-Hydroxy-4-Aminobenzoate. Evidence for a proton channel and a new binding mode of the flavin ring.
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Authors
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H.A.Schreuder,
A.Mattevi,
G.Obmolova,
K.H.Kalk,
W.G.Hol,
F.J.Van der bolt,
W.J.Van berkel.
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Ref.
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Biochemistry, 1994,
33,
10161-10170.
[DOI no: ]
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PubMed id
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Secondary reference #3
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Title
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Crystal structure of the reduced form of p-Hydroxybenzoate hydroxylase refined at 2.3 a resolution.
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Authors
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H.A.Schreuder,
J.M.Van der laan,
M.B.Swarte,
K.H.Kalk,
W.G.Hol,
J.Drenth.
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Ref.
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Proteins, 1992,
14,
178-190.
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PubMed id
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Secondary reference #4
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Title
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Engineering of microheterogeneity-Resistant p-Hydroxybenzoate hydroxylase from pseudomonas fluorescens.
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Authors
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K.Eschrich,
W.J.Van berkel,
A.H.Westphal,
A.De kok,
A.Mattevi,
G.Obmolova,
K.H.Kalk,
W.G.Hol.
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Ref.
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FEBS Lett, 1990,
277,
197-199.
[DOI no: ]
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PubMed id
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Secondary reference #5
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Title
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Crystal structure of the p-Hydroxybenzoate hydroxylase-Substrate complex refined at 1.9 a resolution. Analysis of the enzyme-Substrate and enzyme-Product complexes.
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Authors
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H.A.Schreuder,
P.A.Prick,
R.K.Wierenga,
G.Vriend,
K.S.Wilson,
W.G.Hol,
J.Drenth.
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Ref.
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J Mol Biol, 1989,
208,
679-696.
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PubMed id
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Secondary reference #6
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Title
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The coenzyme analogue adenosine 5-Diphosphoribose displaces FAD in the active site of p-Hydroxybenzoate hydroxylase. An x-Ray crystallographic investigation.
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Authors
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J.M.Van der laan,
H.A.Schreuder,
M.B.Swarte,
R.K.Wierenga,
K.H.Kalk,
W.G.Hol,
J.Drenth.
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Ref.
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Biochemistry, 1989,
28,
7199-7205.
[DOI no: ]
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PubMed id
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Secondary reference #7
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Title
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Crystal structure of p-Hydroxybenzoate hydroxylase complexed with its reaction product 3,4-Dihydroxybenzoate.
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Authors
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H.A.Schreuder,
J.M.Van der laan,
W.G.Hol,
J.Drenth.
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Ref.
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J Mol Biol, 1988,
199,
637-648.
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PubMed id
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Secondary reference #8
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Title
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Crystal structure of p-Hydroxybenzoate hydroxylase.
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Authors
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R.K.Wierenga,
R.J.De jong,
K.H.Kalk,
W.G.Hol,
J.Drenth.
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Ref.
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J Mol Biol, 1979,
131,
55-73.
[DOI no: ]
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PubMed id
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Figure 3.
FIG. 3. The secondary structure of p-OHbz hydroxylase. llBFE indicates the orientation with
respect to Fig. 1. The crystallographic 2.fold axis is indicated. Although not clear from this
diagram, sitlc-chains of H5 ir&ract with side-chaims of Hl. The orientation of FAD is indicated
by 1'1 (isoalloxazie ring), by I' (the phosphate groups) and by Ad (the adenine ring). The gold
(NaAu(CN),) position lia marked by an asterisk. The broken lines delineate the 3 domains. + or
indicate that t,he N-terminal end or the C-terminal end of a /3-strand points to the viewer.
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Figure 10.
FIG. 10. Structure of the oxidized form of the isoalloxazinn ring. The two nitrugen-containing
aromatic rings form the pteridine skeleton.
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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Secondary reference #9
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Title
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Crystallization and preliminary X-Ray investigation of p-Hydroxybenzoate hydroxylase from pseudomonas fluorescens.
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Authors
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J.Drenth,
W.G.Hol,
R.K.Wierenga.
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Ref.
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J Biol Chem, 1975,
250,
5268-5269.
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PubMed id
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