Part:BBa_K5181008

P450_CPR_fusion protein (BC_linker)

This part is an artificial fusion protein. It is a bifunctional self-sufficient p450 that contains the cytochrome p450 monooxygenase domain for CYP736A167 from santalum album and the reductase domain of CPR1 of the same species connected by linker from the naturally existing bifunctional p450 of Bacillus cereus It is meant to catalyze the reaction of converting alpha santalene, beta santalene, epi-beta santalene, and alpha-exo-bergamotene to their respective 'ol' forms by oxidizing them.

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 cereus (and subtilus) to combine the functional domains of both the proteins. The Bifunctional p450 of B.cereus 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) and P450 (BBa_K5181003) of Santalum album and the Bifunctional p450 of Bacillus cereus.

The Green colored Protein represents the Bifunctional P450 of Bacillus cereus, and Cyan represents Truncated P450 and Magenta represents Truncated CPR of Santalum album.

Annotation	Range in basepairs	length (bp)	Misc. info
Truncated p450	1-1410	1410bp	Santalum album p450(CYP736A167) w/o transmembrane domain
Linker	1411-1521	111bp	Bacillus cereus linker (TBD)
Truncated CPR	1522-3384	1836bp	Santalum album CPR1 w/o transmembrane domain and with 6X -His tag at C-terminal end

Annotation

TBD

The structure of our CPR_p450_fusion protein(Bacillus cereus linker) as predicted by alphafold is

The transmembrane tendency of P450-CPR (BC 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 to the membrane. Thus, our fusion protein 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 2686
  Illegal PstI site found at 2965
  
• 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 2686
  Illegal PstI site found at 2965
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2349
  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 2686
  Illegal PstI site found at 2965
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 217
  Illegal PstI site found at 895
  Illegal PstI site found at 2686
  Illegal PstI site found at 2965
  Illegal NgoMIV site found at 346
  
• 1000
  COMPATIBLE WITH RFC[1000]