Part:BBa_K5184004
slCPR2
To equip our insecticide with enhanced prevention efficacy against T. urticae, we also decide to synthesize 9-hydroxy-zingiberene (9HZ) and 9-hydroxy-10,11-epoxy zingiberene (9H10epoZ), two oxidized products of the monocyclic sesquiterpene 7epiZ. In order to make the oxidase ShZPO funciton efficiently, we plan to incorporate the NADPH-cytochrome P450 reductase SlCPR2 as its redox partner and electron supplier. Our exploration of the reductase provide future iGEM team with a novel way of generating sesquiterpenes from a monocyclic sesquiterpene through oxidation carried out by the collaboration of an oxidase and a reductase.
Usage and Biology
SlCPR2 is a NADPH-cytochrome P450 reductase found in Solanum lycopersicum. Analysis of the reductase revealed that their is a membrane anchor domain at the N-terminus of the amino acid sequence of SlCPR2. The enzyme was found to be predominantly localized on the ER membrane. The co-localization of reductase SlCPR2 and oxidase ShZPO on the ER membrane ensures efficient electron transfer. Consisting of two domains, one with a binding site for flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) and another with a binding site for flavin mononucleotide (FMN). SlCPR2 functions through transferring electrons to cytochrome P450 oxidases, in our context ShZPO. To perform its functions, SlCPR2 requires the presence of NADPH and the cofactors FAD and FMN, which are two falvin priteins existing in various redox forms and able to control electron movement. The electrons provided by NADPH are transferred to FAD and FMN in order, and finally, the electrons required for the reduction reaction are transferred. We constructed a novel sesquiterpene synthesis pathway in E. coli. Using glucose as our raw material, we introduce the MVA pathway, which transforms glucose into dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). Afterwards, SltNPPS, a neryl diphosphate synthase catalyze the production of NPP from IPP and DMAPP. Mvan4662 is then introduced to catalyze the formation of Z,Z-FPP. Then, ShZIS transforms Z,Z-FPP into 7epiZ. In the end, ShZPO works collaboratively with SlCPR2 or AtCPR1.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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