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PDBsum entry 2v2e
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Oxidoreductase
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PDB id
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2v2e
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References listed in PDB file
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Key reference
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Title
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The tuberculosis prodrug isoniazid bound to activating peroxidases.
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Authors
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C.L.Metcalfe,
I.K.Macdonald,
E.J.Murphy,
K.A.Brown,
E.L.Raven,
P.C.Moody.
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Ref.
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J Biol Chem, 2007,
283,
6193.
[DOI no: ]
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PubMed id
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Abstract
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Isoniazid (INH, isonicotinic acid hydrazine) is one of only two therapeutic
agents effective in treating tuberculosis. This prodrug is activated by the heme
enzyme catalase-peroxidase (KatG) endogenous to Mycobacterium tuberculosis, but
the mechanism of activation is poorly understood, in part because the binding
interaction has not been properly established. The class I peroxidases ascorbate
peroxidase (APX) and cytochrome c peroxidase (CcP) have very similar active site
structures to KatG, and are also capable of activating isoniazid. We report here
the first crystal structures of complexes of isoniazid bound to APX and CcP.
These are the first structures of isoniazid bound to any activating enzymes. The
structures show that isoniazid binds close to the d-heme edge in both APX and
CcP, although the precise binding orientation varies slightly in the two cases.
A second binding site for INH is found in APX at the g-heme edge close to the
established ascorbate binding site, indicating that the g-heme edge can also
support the binding of aromatic substrates. We also show that in an active site
mutant of sAPX (W41A) INH can bind directly to the heme iron to become an
inhibitor, and in a different mode when the distal histidine is replaced by
alanine(H42A). These structures provide the first unambiguous evidence for the
location of the isoniazid binding site in the class I peroxidases, and provide
rationalisation of isoniazid resistance in naturally occurring KatG mutant
strains of M. tuberculosis.
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Figure 4.
FIGURE 4. Stereo diagrams showing INH bound in the active
site mutants of sAPX W41A and H42A. A, in sAPX·(W41A),
two molecules of INH (brown) are bound in the distal cavity, one
in the same position as sAPX and a second coordinated directly
to the heme iron. The figure shows observed F[o] - F[c]
difference density (in green, contoured at 3  ). B, in sAPX (H42A)
the orientation of INH (brown) is rotated relative to the wild
type and is held in position by a hydrogen bond to Trp-41.
Observed F[o] - F[c] difference density is shown in green. In
both cases the occupancy of the INH is partial and shared with
water molecules that are represented as red spheres.
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Figure 6.
FIGURE 6. Stereo diagram showing INH bound in the ascorbate
binding pocket of sAPX. Hydrogen bonding interactions are
observed between INH and Arg-172 and Lys-31 and via a water to
the propionate group of the heme. There are INH molecules bound
in identical positions in the sAPX (W41A) and sAPX (H42A)
structures. The first INH molecule bound in the distal cavity is
also shown to aid orientation. The protein is shown in green,
the heme group in blue, and INH in pink. Waters are represented
as red spheres. The observed F[o] - F[c] difference density
(contoured at 3 ) for the INH molecules
is shown in green. Figs. 2, 3, 4, 5, 6 were prepared with PyMOL
(22).
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
283,
6193)
copyright 2007.
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