1. Fazeeda Hosein Sarasvati BahadurSingh Nigel Jalsa Cecilia Diaz David Gopaulchan Protein Structure Investigating DFR specificity in anthocyanin biosynthesis

2. 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

3. Simplified diagram of the flavonoid biosynthetic pathway. 3 Malonyl CoA + Coumaroyl CoA aurones flavones isoflavones DFR flavan-4-ols anthocyanin

5. Enzymatic activity of DFR orthologs + represents enzymatic activity for substrate

6. To compare the protein structures of the orthologs of DFR and identify regions that determine enzyme specificity. Objective

7. Methodology

8. 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 Catalytic residues Variable C-terminal region

9. Medicago truncatula DFR1 99 • Medicago truncatula DFR2 88 • Rosa hybrid DFR 50 100 • Fragaria x ananassa DFR 63 • Vitis vinifera DFR • Arabidopsis thaliana DFR2 92 100 • Arabidopsis thaliana DFR • Gerbera hybrid DFR 98 • Petunia x hybrida DFR 99 99 • Ipomoea nil DFR • Anthurium andraeanum DFR • Oryza sativa DFR • Cymbidium hybrid DFR • Iris x hollandica DFR 97 52 • Lilium hybrid DFR 0.05

10. 3D Structure of grapeDFR

11. Petuniasuperimposed on grape DFR

12. Anthuriumsuperimposed on grape DFR

13. 3D model of grapeDFR using ChemBio3D

14. 3D model of AnthuriumDFR using ChemBio3D

15. Comparison of Putative Binding Sites grape Anthurium

16. 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.

17. Conclusion • Two 3D modelling approaches were used: • One based on protein structure homology-modelling, 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.

18. The End

19. Model showing Binding site of Grape DFR (Residues 131-156) • 1

20. 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 • Grape: YFVSK (residues: 163-167) • Ant.. : YFVSK (residues: 163-167)

21. Predicted Binding Site of Anthurium DHF 2

22. 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

23. Comparison of Grape DHF to Anthurium DHF