Part:BBa_K1080008
ChlP
Geranylgeranyl reductase - ChlP encodes a hydrogenase enzyme which catalyses the terminal hydrogenation steps of bacteriochlorophyll biosynthesis [1]. The enzyme, called geranylgeranyl reductase (ChlP or GGR) is responsible for catalysing the hydrogenation of geranylgeranyl diphosphate (GGPP), reducing its’ double bonds to form phytol pyrophosphate, which is required for the synthesis of chlorophylls, carotenoids, quinones and tocopherols [1-3].
Tanaka, et al. [3] showed that by mutating the ChlP gene in transgenic tobacco plants, reducing its activity, both chlorophyll and tocopherol levels are lowered. The transgenic plants had a slower growth rate and a gradually reduced chlorophyll content. They also showed low pigmentation in comparison to the controls.
Operon usage
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.
Protein Structure
The crystal structure of archaeal geranylgeranyl reductase from Sulfolobus acidocaldarius (Sa-GGR) has been determined [4, 5], as visualised in figure 2. Archaeal GGR is involved in the reduction of isoprenoid lipids found in the cell membrane (as opposed to the fatty acyl chains found in Eukarya and Bacteria). The structure and function is comparable to GGR in chlorophyll biosynthesis: it reduces the isoprenoid molecule phytol in the same way; and an NCBI protein BLAST gave the highest similarity between the ChlP protein sequence in Chlamydomonas reinhardtii (the organism from which the ChlP gene was sourced) and S. acidocldarius [4].
Crystal structure of Sa-GGR can be divided into three domains; FAD-binding (green), catalytic (gold), and C-terminal (blue). The FAD-binding and catalytic domains have more conserved structures to other enzymes than the C-terminal domain. Sa-GGR reduces three of the four double bonds in GGPP, resulting in phytol pyrophosphate [4].
PDB accession number: 3ATQ
Figure 1: (A) Structures of chlorophyllide a and bacteriochlorophyllide a. (B) The terminal hydrogenation steps of (bacterio)chlorophyll synthesis, showing gradual reduction of the geranylgeranyl double bonds. R could be either chlorophyllide, bacteriochlorophyllide, or a diphosphate group. [1]
Biobrick Design:
Source Genbank accession: [http://www.ncbi.nlm.nih.gov/nuccore/NW_001843471.1?report=genbank&from=5129848&to=5133289&strand=true NW_001843471.1]
Source Uniprot reference: [http://www.uniprot.org/uniprot/A8HNE8 A8HNE8]
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 64
Amino acid sequence
MVIGGGPSGA CAAETLAKGG VETFLLERKL DNCKPCGGAI PLCMVEEFDL PMEIIDRRVT
KMKMISPSNR EVDVGKTLSE TEWIGMCRRE VFDDYLRNRA QKLGANIVNG LFMRSEQQSA
EGPFTIHYNS YEDGSKMGKP ATLEVDMIIG ADGANSRIAK EIDAGEYDYA IAFQERIRIP
DDKMKYYENL AEMYVGDDVS PDFYGWVFPK YDHVAVGTGT VVNKTAIKQY QQATRDRSKV
KTEGGKIIRV EAHPIPEHPR PRRCKGRVAL VGDAAGYVTK CSGEGIYFAA KSGRMAAEAI
VEGSANGTKM CGEDAIRVYL DKWDRKYWTT YKVLDILQKV FYRSNPAREA FVELCEDSYV
QKMTFDSYLY KTVVPGNPLD DVKLLVRTVS SILRSNALRS VNSKSVNVSF GSKANEERVM
AA
References and documentation are available. Please note the modified algorithm for extinction coefficient.
Number of amino acids: 422
Molecular weight: 47011.7
Theoretical pI: 7.48
Amino acid composition:
Ala (A) 36 8.5%
Arg (R) 28 6.6%
Asn (N) 16 3.8%
Asp (D) 27 6.4%
Cys (C) 9 2.1%
Gln (Q) 9 2.1%
Glu (E) 31 7.3%
Gly (G) 35 8.3%
His (H) 4 0.9%
Ile (I) 24 5.7%
Leu (L) 23 5.5%
Lys (K) 31 7.3%
Met (M) 15 3.6%
Phe (F) 13 3.1%
Pro (P) 17 4.0%
Ser (S) 25 5.9%
Thr (T) 19 4.5%
Trp (W) 4 0.9%
Tyr (Y) 20 4.7%
Val (V) 36 8.5%
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): 58
Total number of positively charged residues (Arg + Lys): 59
Atomic composition:
Carbon C 2069 Hydrogen H 3278 Nitrogen N 574 Oxygen O 628 Sulfur S 24
Formula: C2069H3278N574O628S24 Total number of atoms: 6573
Extinction coefficients:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 52300 Abs 0.1% (=1 g/l) 1.112, assuming all pairs of Cys residues form cystines
Ext. coefficient 51800
Abs 0.1% (=1 g/l) 1.102, 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 42.25 This classifies the protein as unstable.
Aliphatic index: 76.71
Grand average of hydropathicity (GRAVY): -0.368
Design Notes
Incorporated sequence overlap for Gibson assembly and no GC rich region or restriction site in sequence
Source
Chlamydomonas reinhardtii
References
1. Addlesee, H.A., et al., Cloning, sequencing and functional assignment of the chlorophyll biosynthesis gene, chlP, of Synechocystis sp. PCC 6803. FEBS Lett, 1996. 389(2): p. 126-30.
2. Shpilyov, A.V., et al., Inactivation of the geranylgeranyl reductase (ChlP) gene in the cyanobacterium Synechocystis sp. PCC 6803. Biochim Biophys Acta, 2005. 1706(3): p. 195-203.
3. Tanaka, R., et al., Reduced activity of geranylgeranyl reductase leads to loss of chlorophyll and tocopherol and to partially geranylgeranylated chlorophyll in transgenic tobacco plants expressing antisense RNA for geranylgeranyl reductase. Plant Physiol, 1999. 120(3): p. 695-704.
4. Sasaki, D., et al., Structure and mutation analysis of archaeal geranylgeranyl reductase. J Mol Biol, 2011. 409(4): p. 543-57.
5. Kung, Y., et al., Constructing tailored isoprenoid products by structure-guided modification of geranylgeranyl reductase. Structure, 2014. 22(7): p. 1028-36.
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