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

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

DFR OrthologSubstrate dihydrokaempferoldihydroquercetindihydromyricetin Petunia hybrida ++++ Cymbidium Iris Rosa hybrida Oryza sativa Vitis vinifera ++++ Osteospermum hybrida Gerbera hybrida Fragaria ananassa Anthurium andreanum represents enzymatic activity for substrate Enzymatic activity of DFR orthologs

OBJECTIVE 1.To compare the protein structures of the orthologs of DFR and identify regions that determine enzyme specificity.

METHODOLOGY Performing multiple sequence alignmentDeveloping 3D models Mapping regions of variation onto the 3D structures.

Substrate binding site Variable C-terminal region * Catalytic residues 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 * * *

Medicago truncatula DFR1 Medicago truncatula DFR2 Rosa hybrid DFR Fragaria x ananassa DFR Vitis vinifera DFR Arabidopsis thaliana DFR2 Arabidopsis thaliana DFR Gerbera hybrid DFR Petunia x hybrida DFR Ipomoea nil DFR Anthurium andraeanum DFR Oryza sativa DFR Cymbidium hybrid DFR Iris x hollandica DFR Lilium hybrid 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 grapeAnthurium

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 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.

THE END

Model showing Binding site of Grape DFR (Residues ) 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 Grape: YFVSK (residues: ) Ant.. : YFVSK (residues: )

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