Fazeeda Hosein Sarasvati BahadurSingh Nigel Jalsa Cecilia Diaz David Gopaulchan Introduction Anthocyanins are watersoluble vacuolar pigments Occur in all tissues of higher plants eg ID: 775047
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Slide1
Protein Structure
Investigating DFR specificity in anthocyanin biosynthesis
Fazeeda Hosein
Sarasvati BahadurSingh
Nigel Jalsa
Cecilia Diaz
David Gopaulchan
Slide2Introduction
Anthocyanins are water-soluble vacuolar pigmentsOccur in all tissues of higher plants, eg. leaves, stems, roots, flowers, fruits Function in plants - attract pollinators and seed dispersers, protect against harmful UV lightFunction in animals – (Human diet) offer protection against certain cancers, cardiovascular disease and age-related degenerative diseasesUses - food colourings and textile dyes
Anthocyanins in vacuole
Slide33 Malonyl CoA + Coumaroyl CoA
aurones
isoflavones
anthocyanin
flavones
flavan-4-ols
DFR
Simplified diagram of the flavonoid biosynthetic pathway.
Slide4Slide5DFR Ortholog
SubstratedihydrokaempferoldihydroquercetindihydromyricetinPetunia 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
Slide6Objective
To compare the protein structures of the orthologs of DFR and identify regions that determine enzyme specificity.
Slide7Methodology
Slide8Substrate 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 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
*
*
*
Slide9Medicago 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
Slide103D Structure of grape
DFR
Slide11Petunia
superimposed
on grape DFR
Slide12Anthurium
superimposed on grape
DFR
Slide133D model of grape
DFR using ChemBio3D
Slide143D model of
Anthurium
DFR using ChemBio3D
Slide15Comparison of Putative Binding Sites
grape
Anthurium
Slide16Conclusion
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.
Slide17Conclusion
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.
Slide18The End
Slide19Model showing Binding site of Grape DFR (Residues 131-156)
1
Slide20Comparison 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)
Slide21Predicted Binding Site of Anthurium DHF
2
Slide22Substrate 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
Slide23Comparison of Grape DHF to Anthurium DHF