Protein Structure Investigating DFR specificity in anthocyanin biosynthesis Fazeeda Hosein Sarasvati BahadurSingh Nigel Jalsa Cecilia Diaz David Gopaulchan Introduction Anthocyanins are water-soluble vacuolar pigments Occur in all tissues of higher plants, eg. leaves, stems, roots, flowers, fruits
Function in plants - attract pollinators and seed dispersers, protect against harmful UV light Function in animals (Human diet) offer protection against certain cancers, cardiovascular disease and age-related degenerative diseases Uses - food colourings and textile dyes Anthocyanins in vacuole Simplified diagram of the flavonoid biosynthetic pathway. 3 Malonyl CoA + Coumaroyl CoA
+ + represents enzymatic activity for substrate OBJECTIVE 1. To compare the protein structures of the orthologs of DFR and identify regions that determine enzyme specificity. METHODOLOGY Performing multiple sequence alignment Developing 3D models
Mapping regions of variation onto the 3D structures. A. andraeanum Vitis vinifera Rose hybrid F. ananassa M. truncatula Petunia x hybrida Gerbera hybrid Iris x hollandica Lilium hybrid Oryza sativa
C. hybrid Consensus A. andraeanum Vitis vinifera Rose hybrid F. ananassa M. truncatula Petunia x hybrida Gerbera hybrid Iris x hollandica Lilium hybrid Oryza sativa C. hybrid
Consensus * ** A. andraeanum Vitis vinifera Rose hybrid F. ananassa M. truncatula Petunia x hybrida Gerbera hybrid Iris x hollandica
Lilium hybrid Oryza sativa C. hybrid Consensus Substrate binding site Variable C-terminal region * Catalytic residues Medicago truncatula DFR1
99 Medicago truncatula DFR2 88 Rosa hybrid DFR 50 100 63 Fragaria x ananassa DFR Vitis vinifera DFR
3D Structure of grape DFR Petunia superimposed on grape DFR Anthurium superimposed on grape DFR 3D model of grape DFR using ChemBio3D 3D model of Anthurium DFR using ChemBio3D Comparison of Putative Binding Sites
grape Anthurium Conclusion Alignment of the DFR sequences showed high similarity between the DFR orthologs. However the C-terminal was observed to be highly variable suggesting the region may also be involved in substrate specificity. Conclusion Two 3D modelling approaches were used:
One based on protein structure homologymodelling, could not identify potential differences in the substrate-binding regions. The other modelling system based on steric and stereoelectronic factors, potential regions that may be involve in substrate recognition were identified. THE END of Grape DFR (Residues
131-156) 1 Comparison of Grape DHF to Anthurium spp. DHF Grape :TVNIQE-HQLPVYDESCWSDMEFCRAK Ant.. :TVSIHEGRRHL--YDETSWSDVDFCRAK :TV+I E L YDE+ WSD+ +FCRAK Binding site sequence similarity: 57 % Invariant YXXXK motif, feature of the DFR family
Predicted Binding Site of Anthurium DHF 2 Substrate Specificity an Anomaly? For various DFRs, substrate specificity is dependent upon identity of amino acid residue at position 133 If Asparagine - DHK favoured If Aspartic acid - DHQ favoured Comparison of Grape DHF to Anthurium DHF
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