Protein Structure

Investigating DFR specificity in anthocyanin biosynthesis

Fazeeda HoseinSarasvati BahadurSingh

Nigel JalsaCecilia 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-olsDFR

Simplified diagram of the flavonoid biosynthetic pathway.

DFR Ortholog Substrate

dihydrokaempferol dihydroquercetin dihydromyricetin

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 alignment

Developing 3D models

Mapping regions of variation onto the 3D structures.

Substrate binding site

Variable C-terminal region

* Catalytic residues

A. andraeanumVitis viniferaRose hybridF. ananassaM. truncatulaPetunia x hybridaGerbera hybridIris x hollandicaLilium hybridOryza sativaC. hybridConsensus

A. andraeanumVitis viniferaRose hybridF. ananassaM. truncatulaPetunia x hybridaGerbera hybridIris x hollandicaLilium hybridOryza sativaC. hybridConsensus

A. andraeanumVitis viniferaRose hybridF. ananassaM. truncatulaPetunia x hybridaGerbera hybridIris x hollandicaLilium hybridOryza sativaC. hybridConsensus

* * *

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

100

100

99

99

52

97

98

88

50

63

92

99

0.05

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 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 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 familyGrape: YFVSK (residues: 163-167)Ant.. : YFVSK (residues: 163-167)

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 favouredIf Aspartic acid - DHQ favoured

Comparison of Grape DHF to Anthurium DHF