Dijkstra, B.W.

Egmond, M.R.

Steiner, R.A.

University of Groningen

Structural basis for the oxygenation of flavonols by flavonol 2,3-dioxygenase

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Structural basis for the oxygenation of flavonols by flavonol 2,3-dioxygenase.
Steiner, R.A.; Egmond, M.R.; Dijkstra, B.W.
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Publication date:
2000
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Citation for published version (APA):
Steiner, R. A., Egmond, M. R., & Dijkstra, B. W., (2000). Structural basis for the oxygenation of flavonols by
flavonol 2,3-dioxygenase., 1 p.
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s8a.m1.o3 Structural basis for the oxygenation of
flavonols by flavonol 2,3-dioxygenase. R.A. Steiner#, P.I
van Noort¶, M.R. Egmond¶ and  B. W. Dijkstra# #
Department of Biophysical Chemistry, University of
Groningen Nijenborgh 4, 9747 AG Groningen,  the
Netherlands¶ Unilever Research Laboratory Olivier van
Noortlaan 120, 3133 AT Vlaardingen, the Netherlands
Keywords: enzyme catalysis, protein engineering.
Dioxygenases are enzymes that catalyse the
incorporation of both oxygen atoms of molecular oxygen
into the substrate.
O2 mainly due to its triplet (
3Σg) ground state possesses
under physiological conditions a low kinetic reactivity
towards organic compounds which generally exhibit a
singlet fundamental state. In order to circumvent the spin
selection rule biological system have evolved several
pathways. Complexion to a transition metal is a method
often employed as activation route. Iron, in both haem and
non-haem forms, is the co-factor commonly found in
dioxygenases.
Flavonol 2,3 dioxygenase (FDO) from Aspergillus
japonicus is unique among dioxygenases because it
contains only one cupric copper ion per molecule and no
other co-factors1.
FDO catalyses the oxidation of flavonols (3-hydroxy
flavones) to yield carbon monoxide and the relative
depside (phenolic carboxylic acid ester). Since FDO has
been reported for the first time in the degradation pathway
of quercetin (3,5,7,3’,4’-pentahydroxy flavone) it is also
known as Quercetinase.
Relevant to FDO are the two classes of iron
dioxygenases (intradiol and extradiol dioxygenases)
containing non-haem iron as sole co-factor. It has been
proposed that intradiol dioxygenases activate the metal
bound substrate whilst the extradiol type activates the
dioxygen bound to the ferrous ion2. In all the postulated
mechanisms for non-heam iron dioxygenases indicate,
anyway, a direct co-ordination of molecular oxygen to the
metal centre at some stage of the process.
Anaerobic and aerobic structural studies suggest a
possible reaction mechanism for this peculiar enzyme.
[1] Oka, T. and Simpson, F.J. (1971). Quercetinase, a dioxygenase
containing copper. Biochem and Biophys. Research Comm. 43, 1-5.
[2] Howard, J.B. and Rees, D.C. (1991). Perspectives on non-heme
protein chemistry. Advan. Protein Chem. 42, 199-280.
s8a.m1.o4 Crystallographic studies of the interaction
between the Ferredoxin:NADP+:Reductase and
Ferredoxin R. Morales, M.H. Charon, M. Frey, LCCP,
Institut de Biologie Structurale J.P. Ebel,CEA-CNRS,41
rue J. Horowitz 38027 Grenoble.
Keywords: enzyme catalysis, protein engineering.
Ferredoxin:NADP+:Reductase (FNR) catalyses one
terminal step of conversion of light energy into chemical
energy during photosynthesis. FNR uses two
photoproduced high energy electrons conveyed, one by
one, from the photosystem I by a ferredoxin (Fd) to
catalyse the production of NADPH. Electron transfer
between FNR and Fd requires the formation of a ternary
NADP+/FNR/Fd complex1.
We have solved the structure of a crystallographic
complex between Fd and FNR from the cyanobacterium
Anabaena PCC7119 at 2.4 Å resolution. This gave the first
three-dimensional picture of a Fd/FNR biologically
relevant complex.
The crystal cell parameters are a=b= 63.72 Å and c=
158.02 Å ; space group P212121.
The asymmetric unit contains two FNR (FNR1 and
FNR2, molecular weight, mw, :2×35 kDa) and one
Ferredoxin (mw :11 kDa) molecules. The packing of the
FNR molecules displays a nearly tetragonal symmetry
(S.G. P43212) whereas the Fd arrangement is orthorhombic
( S.G. P212121).
For the computation, the crystal was treated as a
merohedral twin with two components related by a [110]
dyad axis. This approach proved to be a very powerful tool
to locate this elusive ferredoxin and to obtain fully
interpretable electron density maps.
[1] Batie C. "FERREDOXIN:NADP+ OXIDASE Equilibria in Binary
and Ternary Complexes with NADP+ and Fd.", J. Biol. Chem, (1984),
259: 8832 - 8839.
