Coding
DVR1

Part:BBa_K1080012

Designed by: Macquarie University   Group: iGEM13_Macquarie_Australia   (2013-09-22)

DVR1


3,8-divinyl protochlorophyllidea 8-vinyl reductase - Predicted chloroplast transit peptide 1-58; [PMID: 15695432; 15849308] This enzyme reduces divinyl protochlorophyllide a to monovinyl protochlorophyllide a. This reduction is essential for chlorophyllide a synthesis, but can probably occur at any stage between protoporphyrin IX and chlorophyllide a. [1, 2]

Usage and Biology

DVR1 reaction image.png
Figure 1: Reaction catalysed by DVR1. [3]

Operon usage


PSB1C3 Operon 3.png
This gene has been used in an operon with other genes responsible for the terminal steps of the chlorophyll biosynthesis pathway, in the conversion of divinyl protochlorophyllide to chlorophyll a. DVR1 reduces divinyl protochlorophyllide, POR converts protochlorophyllide to chlorophyllide, ChlG adds the geranylgeranyl pyrophosphate chain to the chlorophyllide molecule, and ChlP reduces the double bonds on GGPP. The final product is chlorophyll a.
The plasmid is under the control of the lac promoter.

Biobrick Design:

Source Genbank accession: [http://www.ncbi.nlm.nih.gov/nuccore/NW_001843471.1?report=genbank&from=4166415&to=4170852&strand=true NW_001843471.1]

Source Uniprot reference: [http://www.uniprot.org/uniprot/A8HMQ3 A8HMQ3]

cDNA gene sequence from Chlamydomonas reinhardtii was sourced from NCBI database, chloroplast targeting sequence was removed. EcoRI/XbaI/SpeI/PstI restriction sites were removed via codon adjustment, biobrick prefix and RBS were added to start of gene, biobrick suffix added to end of gene.

Biobrick construction: Gibson assembly of 2 synthesised DNA fragments into BB vector.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 288
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]




Amino acid sequence

MAMAASRQAV RVAAAVDADY RKREPKDVRV LVVGPTGYIG KFVVKELVSR GYNVVAFARE
NAGIKGKMGR EDIVKEFHGA EVRFGSVLDP ASLRDVAFKD PVDVVVSCLA SRTGGKKDSW
LIDYTATKNS LDVARASGAK HFVLLSAICV QKPLLEFQKA KLQFESDLQA AGDITYSIVR
PTAFFKSIAG QIDIVKKGNP YVMFGDGNLA ACKPISEADL ASFIADCVTE QNKVNKVLPI
GGPSKAFTAK QQADLLFNIT GLPPKYFPVP VALMDGMIGL FDSLAKLFPQ LEDSAEFARI
GKYYATESML VYDEARGVYR KTKRLVTART RWKTSSLVQ

References and documentation are available. Please note the modified algorithm for extinction coefficient.

Number of amino acids: 339

Molecular weight: 37034.9

Theoretical pI: 9.40

Amino acid composition:
Ala (A) 40 11.8%
Arg (R) 19 5.6%
Asn (N) 8 2.4%
Asp (D) 22 6.5%
Cys (C) 4 1.2%
Gln (Q) 11 3.2%
Glu (E) 14 4.1%
Gly (G) 24 7.1%
His (H) 2 0.6%
Ile (I) 16 4.7%
Leu (L) 27 8.0%
Lys (K) 29 8.6%
Met (M) 7 2.1%
Phe (F) 18 5.3%
Pro (P) 15 4.4%
Ser (S) 21 6.2%
Thr (T) 14 4.1%
Trp (W) 2 0.6%
Tyr (Y) 11 3.2%
Val (V) 35 10.3%
Pyl (O) 0 0.0%
Sec (U) 0 0.0%

(B)   0	  0.0%
(Z)   0	  0.0%
(X)   0	  0.0%


Total number of negatively charged residues (Asp + Glu): 36 Total number of positively charged residues (Arg + Lys): 48

Atomic composition:

Carbon C 1670 Hydrogen H 2668 Nitrogen N 450 Oxygen O 477 Sulfur S 11

Formula: C1670H2668N450O477S11 Total number of atoms: 5276

Extinction coefficients:

Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.

Ext. coefficient 27640 Abs 0.1% (=1 g/l) 0.746, assuming all pairs of Cys residues form cystines


Ext. coefficient 27390 Abs 0.1% (=1 g/l) 0.740, assuming all Cys residues are reduced

Estimated half-life:

The N-terminal of the sequence considered is M (Met).

The estimated half-life is: 

                            30 hours (mammalian reticulocytes, in vitro).
                           >20 hours (yeast, in vivo).
                           >10 hours (Escherichia coli, in vivo).


Instability index:

The instability index (II) is computed to be 31.02 This classifies the protein as stable.


Aliphatic index: 91.21

Grand average of hydropathicity (GRAVY): -0.019

Source

Chlamydomonas reinhardtii,

References

1. Willows, R.D., Chlorophylls, in Plant Pigments and their Manipulation, K. Davies, Editor. 2004, Blackwell Publishing.
2. Willows, R.D., Chlorophylls, in Encyclopedia of plant and crop science. 2004.
3. DiatomCyc. Chlamydomonas reinhardtii Reaction: 1.3.1.75. Available from: http://akongo.psb.ugent.be/CHLAMY/NEW-IMAGE?type=REACTION&object=RXN1F-72.



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