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PDBsum entry 3hgo
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Oxidoreductase
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PDB id
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3hgo
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References listed in PDB file
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Key reference
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Title
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Structural basis of substrate specificity of plant 12-Oxophytodienoate reductases.
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Authors
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C.Breithaupt,
R.Kurzbauer,
F.Schaller,
A.Stintzi,
A.Schaller,
R.Huber,
P.Macheroux,
T.Clausen.
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Ref.
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J Mol Biol, 2009,
392,
1266-1277.
[DOI no: ]
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PubMed id
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Abstract
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12-Oxophytodienoate reductase 3 (OPR3) is a FMN-dependent oxidoreductase that
catalyzes the reduction of the cyclopentenone (9S,13S)-12-oxophytodienoate
[(9S,13S)-OPDA] to the corresponding cyclopentanone in the biosynthesis of the
plant hormone jasmonic acid. In vitro, however, OPR3 reduces the jasmonic acid
precursor (9S,13S)-OPDA as well as the enantiomeric (9R,13R)-OPDA, while its
isozyme OPR1 is highly selective, accepting only (9R,13R)-OPDA as a substrate.
To uncover the molecular determinants of this remarkable enantioselectivity, we
determined the crystal structures of OPR1 and OPR3 in complex with the ligand
p-hydroxybenzaldehyde. Structural comparison with the OPR1:(9R,13R)-OPDA complex
and further biochemical and mutational analyses revealed that two active-site
residues, Tyr78 and Tyr246 in OPR1 and Phe74 and His244 in OPR3, are critical
for substrate filtering. The relatively smaller OPR3 residues allow formation of
a wider substrate binding pocket that is less enantio-restrictive. Substitution
of Phe74 and His244 by the corresponding OPR1 tyrosines resulted in an OPR3
mutant showing enhanced, OPR1-like substrate selectivity. Moreover, sequence
analysis of the OPR family supports the filtering function of Tyr78 and Tyr246
and allows predictions with respect to substrate specificity and biological
function of thus far uncharacterized OPR isozymes. The discovered structural
features may also be relevant for other stereoselective proteins and guide the
rational design of stereospecific enzymes for biotechnological applications.
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Figure 2.
Fig. 2. Binding of PHB to OPR1 and OPR3. (a) 2F[o ]− F[c]
omit electron density map of the complex structures of OPR1:PHB
(left) and OPR3:PHB (right), at 2.30- and 2.07-Å
resolution, contoured at 1.0σ. For map calculation, PHB was
omitted from the model. (b) Stereo view of the superposition of
the active-site cavities of OPR1:PHB (light blue) and
OPR1:(9R,13R)-OPDA (green). The PHB ligand is shown in dark blue
and OPDA in yellow. In addition, the ribbon structure of OPR1 is
shown. (c) Stereo view of the superposition of the active-site
cavities of OPR1:PHB (light blue), OPR3:PHB (yellow), and the
OPR3 double mutant OPR3YY (green). The PHB ligand is shown in
dark blue (OPR1) and orange (OPR3). In addition, the ribbon
structure of OPR3 is shown. Residues are numbered according to
the OPR3 sequence.
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Figure 3.
Fig. 3. Substrate binding to OPR1 and OPR3. (a) Stereo view
of the active-site cavity of the OPR1:(9R,13R)-OPDA complex. In
addition to the molecular surfaces of OPR1 (grey; surface of
FMN: green) and of the substrate (9R,13R)-OPDA (yellow), Tyr246
and Tyr78 that narrow the opening of the cavity as well as
(9R,13R)-OPDA are shown as ball-and-stick models (blue). (b)
Stereo view of the active-site cavity of a modeled
OPR3:(9R,13R)-OPDA complex. Surfaces were colored as in (a). The
complex was obtained by transferring the substrate's coordinates
of the aligned OPR1:(9R,13R)-OPDA complex to the OPR3 structure.
Protein residues of OPR3 and the substrate's carboxy alkyl chain
(shortened in the figure for clarity) clash in the model because
OPR3 lacks the tunnel that accommodates the carboxy alkyl chain
in OPR1. In OPR3, these clashes can be easily avoided by a
change in the conformation of atoms C1 to C8 of the carboxy
alkyl chain. In comparison to OPR1, the opening of the OPR3
cavity is lined by His244 and Phe74, resulting in a wider
entrance and leaving more space near the stereo centers of the
substrate.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2009,
392,
1266-1277)
copyright 2009.
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