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 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. 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

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 family

Grape: 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 favoured

If Aspartic acid - DHQ favoured

Comparison of Grape DHF to

Anthurium DHF