Part:BBa_K4462110
pCAM
Construct for constitutive expression of the Cytochrome P450cam enzyme system
Usage and Biology
The Cytochrome P450cam enzyme system is present naturally in many strains of Pseudomonas putida with its fundamental role being camphor degradation. The fundamental motivation for using this enzyme for halocarbon degradation lies in the fact that this enzyme can act naturally as an oxidant but can act as a reductant at times, this reducing property is what motivated us to research more about this enzyme. It is a heterotrimeric protein consisting of a Cytochrome P450 enzyme, a Putidaredoxin complexed with cofactors 2Fe-2S, and an NADH-dependent Putidaredoxin reductase. CamC codes for the Cytochrome P450 enzyme while CamA codes for the NADH dependant Putaredoxin reductase and CamB codes for the Putidaredoxin. 20-37 degree Celsius and pH 6-8 are the most optimum conditions for the activity of this enzyme.
It works under both aerobic and anaerobic conditions but gives different products with halocarbons. Under anaerobic conditions, it seems to remove vicinal halogens and form a double bond. This characteristic degradation seems to work only when at least one of the two vicinal halogens is chlorine, bromine or iodine. It doesn't seem to degrade halocarbons with one carbon and halocarbons with all vicinal halogen combinations including two fluorine atoms. The removal of vicinal halogens is based on size i.e larger halogens are removed more easily. Under aerobic conditions, it gives an oxidized product rather than a reduced one under anaerobic conditions. Existing observations, showcase that under aerobic conditions, Cytochrome P450 cam converts the carbon position with lesser number of halogens or weaker halogens into a COOH group.
The plasmid pCAM was designed to expresses the three-component Cytochrome P450cam enzyme system as a reconstituted operon under the control of the strong constitutive EM7 promoter (pCAM7) or the 2-methylbenzoate inducible XylS/Pm promoter (pCAM5). The operon as designed consists of the CamC, CamA, and CamB genes and was supposed to be inserted either into the pSEVA438 (medium-copy, streptomycin-resistance, XylS/Pm regulator) or into pSEVA2213 (medium-copy, kanamycin-resistance, EM7 promoter).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1709
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2062
- 1000COMPATIBLE WITH RFC[1000]
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