Coding

Part:BBa_K5181016

Designed by: Eeshani Umesh Abhyankar   Group: iGEM24_IISER-TVM   (2024-09-11)


P450_CPR fusion protein(BM linker)

This part is an artificial fusion protein. It is composed of the truncated cytochrome P450 monooxygenase domain from CYP736A167 and truncated reductase domain of CPR1 from santalum album connected by BM linker from the naturally existing bifunctional P450 of Bacillus megaterium. It is meant to catalyze the reaction of oxidising alpha santalene, beta santalene, epi-beta santalene, and alpha-exo-bergamotene to their respective 'ol' forms.

Usage and Biology

The Rationale of the Design: To tackle the problem of expression of transmembrane proteins in prokaryotic systems and Co-localization of CPR and P450 to be functionally active, we came up with a design plan to truncate the transmembrane domain of Both CPR and P450 and follow the architecture of a soluble, naturally existing bifunctional p450 present in Bacillus megaterium to combine the functional domains of both the proteins. The Bifunctional p450 of B.megaterium was chosen because of the stark structural similarities between the proteins despite having low sequence similarity as exhibited by their alphafold structures. The following picture was generated by superimposing the Truncated CPR (BBa_K5181001), truncated P450 (BBa_K5181003) and BM linker (BBa_K5181012) of Santalum album and the Bifunctional P450 of Bacillus megaterium.

Predicted P450-CPR fusion protein (BM linker) structure


The linear map of fusion protein is depicted below.

Map

The transmembrane tendency of P450-CPR (BM linker) fusion protein


The extreme N-terminal and C-terminal residues show low transmembrane(below 0) tendencies, and thus, we can presume the protein will not get embedded in the membrane. Thus, it's more likely to behave like certain bifunctional P450s found in prokaryotes.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 217
    Illegal PstI site found at 895
    Illegal PstI site found at 2665
    Illegal PstI site found at 2944
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 370
    Illegal PstI site found at 217
    Illegal PstI site found at 895
    Illegal PstI site found at 2665
    Illegal PstI site found at 2944
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2328
    Illegal BamHI site found at 3364
    Illegal XhoI site found at 1306
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 217
    Illegal PstI site found at 895
    Illegal PstI site found at 2665
    Illegal PstI site found at 2944
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 217
    Illegal PstI site found at 895
    Illegal PstI site found at 2665
    Illegal PstI site found at 2944
    Illegal NgoMIV site found at 346
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None