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PDBsum entry 1knh
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References listed in PDB file
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Key reference
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Title
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Molecular basis for the stabilization and inhibition of 2, 3-Dihydroxybiphenyl 1,2-Dioxygenase by t-Butanol.
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Authors
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F.H.Vaillancourt,
S.Han,
P.D.Fortin,
J.T.Bolin,
L.D.Eltis.
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Ref.
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J Biol Chem, 1998,
273,
34887-34895.
[DOI no: ]
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PubMed id
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Abstract
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The steady-state cleavage of catechols by 2,3-dihydroxybiphenyl 1, 2-dioxygenase
(DHBD), the extradiol dioxygenase of the biphenyl biodegradation pathway, was
investigated using a highly active, anaerobically purified preparation of
enzyme. The kinetic data obtained using 2,3-dihydroxybiphenyl (DHB) fit a
compulsory order ternary complex mechanism in which substrate inhibition occurs.
The Km for dioxygen was 1280 +/- 70 microM, which is at least 2 orders of
magnitude higher than that reported for catechol 2,3-dioxygenases. Km and Kd for
DHB were 22 +/- 2 and 8 +/- 1 microM, respectively. DHBD was subject to
reversible substrate inhibition and mechanism-based inactivation. In
air-saturated buffer, the partition ratios of catecholic substrates substituted
at C-3 were inversely related to their apparent specificity constants. Small
organic molecules that stabilized DHBD most effectively also inhibited the
cleavage reaction most strongly. The steady-state kinetic data and
crystallographic results suggest that the stabilization and inhibition are due
to specific interactions between the organic molecule and the active site of the
enzyme. t-Butanol stabilized the enzyme and inhibited the cleavage of DHB in a
mixed fashion, consistent with the distinct binding sites occupied by t-butanol
in the crystal structures of the substrate-free form of the enzyme and the
enzyme-DHB complex. In contrast, crystal structures of complexes with catechol
and 3-methylcatechol revealed relationships between the binding of these smaller
substrates and t-butanol that are consistent with the observed competitive
inhibition.
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Figure 3.
Fig. 3. Relationship of the two t-butanol-binding sites
to groups at the active site. A, in the free enzyme, t-butanol
occupies the distal portion of the substrate-binding site. B, in
the enzyme-substrate complexes, t-butanol occupies an auxiliary
site adjacent to the distal portion of the substrate-binding
site, further removed from the iron.
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Figure 4.
Fig. 4. Electron density maps and models illustrating the
structure of the DHBD:3-methylcatechol-bound and substrate-free
forms of DHBD. Each part is a (divergent) stereo drawing
prepared with the program MolView (42). The identical observed
structure factors were used in all maps, which demonstrates the
presence of both forms of the enzyme in the same crystal. All
maps are at 1.9-Å resolution and are contoured at two
standard deviations above the mean of the map. In the models,
the carbon atoms are more darkly shaded than the nitrogen and
oxygen atoms. A, F[o]  F[c]
electron density representing the iron, 3-methylcatechol, and
two water ligands. The F[c] 's and phases are from the
structure of the substrate-free enzyme (3). The model is the
initial fit to this density. B, residual F[o] F[c]
electron density following refinement of a model that included
the iron, 3-methylcatechol, two water ligands, and a t-butanol
bound in the auxiliary site, as shown. The F[c] 's and phases
are from this model. The density features arise from the
fraction of the crystal that is in the substrate free-form, as
demonstrated by C, which shows the refined model of this form
(3) in conjunction with the same density. Note that the
t-butanol binds in the substrate-binding site in this form of
the enzyme.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(1998,
273,
34887-34895)
copyright 1998.
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