Crystal structure of grape dihydroflavonol 4-reductase, a key enzyme in flavonoid biosynthesis.
The nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enzyme dihydroflavonol 4-reductase (DFR) catalyzes a late step in the biosynthesis of anthocyanins and condensed tannins, two flavonoid classes of importance to plant survival and human nutrition. This enzyme has been widely investigated in many plant species, but little is known about its structural and biochemical properties. To provide a basis for detailed structure-function studies, the crystal structure of Vitis vinifera DFR, heterologously expressed in Escherichia coli, has been determined at 1.8 A resolution. The 3D structure of the ternary complex obtained with the oxidized form of nicotinamide adenine dinucleotide phosphate and dihydroquercetin, one of the DFR substrates, presents common features with the short-chain dehydrogenase/reductase family, i.e., an N-terminal domain adopting a Rossmann fold and a variable C-terminal domain, which participates in substrate binding. The structure confirms the importance of the 131-156 region, which lines the substrate binding site and enlightens the role of a specific residue at position 133 (Asn or Asp), assumed to control substrate recognition. The activity of the wild-type enzyme and its variant N133D has been quantified in vitro, using dihydroquercetin or dihydrokaempferol. Our results demonstrate that position 133 cannot be solely responsible for the recognition of the B-ring hydroxylation pattern of dihydroflavonols.
Petit
Pierre
P
Chimie et Biologie des Membranes et des Nanoobjets, UMR CNRS 5248, Bâtiment B8, Avenue des Facultés, Université Bordeaux 1, 33405 Talence Cedex, France.
Granier
Thierry
T
d'Estaintot
Béatrice Langlois
BL
Manigand
Claude
C
Bathany
Katell
K
Schmitter
Jean-Marie
JM
Lauvergeat
Virginie
V
Hamdi
Saïd
S
Gallois
Bernard
B
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005419:Flavonoids / Q000096:biosynthesis*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D027843:Vitis / Q000201:enzymology*
D005419:Flavonoids; D010940:Plant Proteins; D009249:NADP; D000429:Alcohol Oxidoreductases; C056570:dihydroflavanol 4-reductase