Development of a new platform technology for plant Cytochrome P450 fusions

To date more than 15000 Cytochromes P450 have been identified and named so far, with one third belonging to the plant kingdom. This is a key biochemical resource, providing a wealth of biocatalysts covering a diverse range of chemistries. Characterisation, however, has been greatly hindered by the poor solubility of many P450s, a result of the membrane anchoring region common to all plant P450s. Fusions of plant P450 heme domains to an appropriate reductase without the hydrophobic membrane anchor could provide the basis for developing robust, soluble plant enzyme systems for substrate screens to discover novel activities that are also of benefit to industry.
In this project, the two predominantly expressed Arabidopsis reductases ATR1 and ATR2 have been cloned, without the membrane anchor, and expressed in Escherichia coli. These two truncated enzymes have been purified and assessed for activity with ATR2 found to be more active than ATR1. ATR2 was chosen for engineering into a novel plant P450 reductase vector platform for high throughput applications, whereby the P450s can be easily and quickly swapped using ligation independent cloning techniques. Four different plant P450s (CYP93C1, CYP73A5, CYP82E4 and CYP81D8) were selected to validate this technology, and activity for the fusions of CYP93C1 (Isoflavone synthase I from Glycine max) and CYP73A5 (cinnamate-4-hydroxylase from Arabidopsis) with ATR2 have been shown. The presence of CYP73A5 fused to ATR2 was verified through purification and further studies showed that it has to be membrane associated for activity.
Additionally, CYP93C1 and CYP73A5 were also fused with the bacterial RhF reductase from Rhodococcus sp. and expressed in E. coli and compared to the plant P450 – plant reductase fusion protein. These novel plant-bacterial fusion P450 systems are the first example of active plant P450s fused to a reductase from a bacterium.
This platform technology will provide the possibility for characterisation studies of eukaryotic P450s with unknown function and the discovery of new activities.