Dihydroflavonol 4-reductase (DFR) is a key enzyme within anthocyanin biosynthesis known for its distinct substrate specificity found in various plant species (1,2). Based in the findings of enzymatic studies and in vivo inhibition experiments in cyanidin accumulating genotype “Clivia” two DFRs with different substrate preference were postulated. Cloning approaches with petals of “Clivia” led to the identification of a second DFR sequence sharing around 96% identity to the previously cloned DFR from pelargonidin accumulating variety “Regina” (1,3). The obtained recombinant protein shows a higher preference for dihydroquercetin (DHQ) while dihydrokaempferol (DHK) was still converted to leucopelargondin (LPg) to a certain extend. However, the overall ratio showed a clear preference for the cyaniding branch of the pathway. To shed light on this biochemical aspect of the anthocyanins biosynthetic pathway, we have attempted an in silico structure-based approach aiming to relate the specific amino acid differences in the DFRs of Gerbera “Regina” and “Clivia” varieties, with their molecular structures and enzyme selectivity data. The DFR models have been built using the crystal structure of the DFR from Vitis vinifera4 (PDB ID 2C29) as a template. We suggest that a residue belonging to the “specificity loop” located near the substrate “binding site” confers the observed substrate-binding specificity. Namely, Gly135 (Gerbera “Regina” DFR) or Val135 (Gerbera “Clivia” DFR) respectively, is likely to unlock or lock the orientation of the conserved residue Asn134 that in turn is engaged in hydrogen bonding interactions with 4’ OH’ of ring B of DHK or both 3’ OH’ and 4’ OH’ of ring B of DHQ. Interestingly the side chain orientation of Val135 (Gerbera “Clivia” DFR) is restricted and stabilized by hydrophobic interactions with the conserved residues Ile154 and Phe165.
|Citation:||Cassetta, A.; Lamba, D.; Bashandy, H.; Teeri, T.H.; Martens, S. (2013). Structural analysis of two distinct dihydroflavonol 4-reductases in Gerbera Hybrids. In: 7th International Workshop on Anthocyanins, Porto, Portugal, September 9-11, 2013. url: http://iwa2013.com/home/ handle: http://hdl.handle.net/10449/22863|
|Organization unit:||Food Quality and Nutrition Department # CRI_2011-JAN2016|
|Authors:||Cassetta, A.; Lamba, D.; Bashandy, H.; Teeri, T.H.; Martens, S.|
|Title:||Structural analysis of two distinct dihydroflavonol 4-reductases in Gerbera Hybrids|
|Appears in Collections:||03 - Conference object|