Alteration of a single amino acid changes the substrate specificity of dihydroflavonol 4-reductase

Many plant species exhibit a reduced range of flower colors due to the lack of an essential gene or to the substrate specificity of a biosynthetic enz yme. Petunia does not produce orange flowers because dihydroflavonol 4-redu ctase (DFR) from this species, an enzyme involved in anthocyanin biosynthes is, inefficiently reduces dihydrokaempferol, the precursor to orange pelarg onidin-type anthocyanins. The substrate specificity of DFR, however, has no t been investigated at the molecular level. By analyzing chimeric DFRs of P etunia and Gerbera, we identified a region that determines the substrate sp ecificity of DFR. Furthermore, by changing a single amino acid in this pres umed substrate-binding region, we developed a DFR enzyme that preferentiall y reduces dihydrokaempferol. Our results imply that the substrate specifici ty of DFR can be altered by minor changes in DFR.