Part:BBa_K3114027
Pheophytinase (PPH)
Usage and Biology
Pheophytinase is a chloroplast-located hydrolase which dephytylates the pigment pheophytin during the chlorophyll degradation pathway in plants and algae (Schelbert et al., 2009). Pheophytinase catalyzes the reaction removing the phytyl chain from pheophytin resulting in the formation of pheophorbide a. In several plant species, it has been shown that absence of pheophytinase results in a stay-green phenotype (Guyer et al., 2018).
BBa_K3114024 is a part consisting of multiple components of iGEM Calgary's 2019 Biobrick submission kit as well as other widely used Biobricks. The coding region consists of the PPH gene which encodes for pheopytinase(BBa_K3114011).A T7 promoter(BBa_I719005)was used in conjunction with a strong ribosome binding site(BBa_B0030).Following the coding region is iGEM Calgary's universal spacer sequence and 6XHis tag site(BBa_K3114014).The universal spacer was utilized in an attempt to mitigate any adverse effects that the his tag may have on the protein's function, as hypothesized in Meguro et al. 2011.
This part can be used for IPTG-inducible expression and secretion of the protein pheophytinase. This circuit contains the DsbA signal peptide. The DsbA signal peptide is a 19-amino acid sequence which targets fused proteins to the Sec secretion pathway in E. coli (Schierle et al., 2003). The fused protein is not folded until it is secreted to the periplasm. The signal peptide is cleaved after residue 19 by signal peptidase after secretion (recognition sequence: Ala-X-Ala).
His Tag Purification
Meguro et al. 2011 purified 7-HCAR using a his-tag/nickel column, but the recombinant protein did not exhibit enzymatic activity. The authors conjectured that the "His tag at the N terminus disturbed the HCAR activity" (Meguro et al. 2011).
The 2019 Calgary iGEM team set out to get around this problem. The team modelled the electrostatic interactions of 7-HCAR and discovered that 7-HCAR has a highly electronegative core, which is likely interfering with the positively charged His tag. iGEM Calgary developed a universal spacer sequence (https://parts.igem.org/Part:BBa_K3114014) which permitted successful His tag purification.
Sequences and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1579
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 267
Illegal AgeI site found at 558 - 1000COMPATIBLE WITH RFC[1000]
References
Luzia Guyer, Kathrin Salinger, Undine Krügel, Stefan Hörtensteiner, Catalytic and structural properties of pheophytinase, the phytol esterase involved in chlorophyll breakdown, Journal of Experimental Botany, Volume 69, Issue 4, 6 February 2018, Pages 879–889, https://doi.org/10.1093/jxb/erx326.
Meguro, M., Ito, H., Takabayashi, A., Tanaka, R., & Tanaka, A. (2011). Identification of the 7-hydroxymethyl chlorophyll a reductase of the chlorophyll cycle in Arabidopsis. The Plant Cell, 23(9), 3442-3453.
Schelbert, S., Aubry, S., Burla, B., Agne, B., Kessler, F., Krupinska, K., & Hörtensteiner, S. (2009). Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis. The Plant Cell, 21(3), 767-785.
Schierle, C. F., Berkmen, M., Huber, D., Kumamoto, C., Boyd, D., & Beckwith, J. (2003). The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway. Journal of Bacteriology, 185(19), 5706–5713. https://doi.org/10.1128/JB.185.19.5706-5713.2003
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