Re-investigating substrate specificity of dihydroflavonol 4-reductase with respect to the B-ring hydroxylation pattern of substrates

Dihydroflavonol 4-reductase (EC 1.1.1.219, DFR) catalyses the reduction of (2R,3R)-(+)-dihydroflavonols into (2R,3S,4S)-cis-3,4-leucoanthocyanidins. Depending on the plant species, DFR can be unspecific with regard to the B-ring hydroxylation pattern or selective, as in Petunia hybrida in which the DFR does not convert DHK, or in Fragaria species where a pair of DFRs are present that shows contrasting substrate specificity with regard to DHK. DFR substrate specificity has been largely investigated in many plant species. The amino acids determining DFR substrate specificity are not yet completely understood, but previous studies have identified a region of 26 amino acids putatively relevant and in particular, an aspartic acid in position 134, that seems to be responsible for the non-acceptance of DHK as substrates. The recently identified pair of Fragaria DFRs with contrasting substrate specificity was used to study putative regions responsible for the divergent substrate specificity. We demonstrate that neither the versatile C-terminus nor the DFR length nor two of three putative regions are of any relevance. In addition, we analyse previously published DFRs of Malus × domestica, Pyrus communis and Ginkgo biloba and the correlation between their substrate specificity and amino acid sequences. Technical constraints of DFR enzyme assays and potential putative substrate specificity bias is discussed.