Design, synthesis and application of novel cytochrome P450 probes

Almost fifty years ago, cytochrome P450s (P450) in animal cells were discovered. Several years later, it was reported that these enzymes possessed the characteristics of a heme-containing protein (like those preent in red blood cells) but they were distinct from another cytochrome enzyme, that of cytochrome b5. This report named these new substances P450 (P for pigment). Concurrently, other research had demonstrated that certain oxygen and NADPH-dependent microsomal enzymes had the ability to catalyse a myriad of reactions including hydroxylation, deamination, oxidation and N-dealkylation. Subsequent research was able to link the now labelled P450s with the enzymatic function described by the concurrent research. As a result, it soon became apparent that the P450 enzymes represented a major class of proteins that we now refer to as cytochrome P450-dependent monooxygenases. Subsequently, these enzymes have been found in bacteria, insects, plants, fish, mammals, and fungi. As a direct result of their importance, particularly in drug and xenobiotic metabolism, a great deal of research has been conducted into the roles, identification of the sequences and the catalytic mechanisms of animal and bacterial P450s. Plant P450s on the other hand have been largely neglected and were only positively identified fifteen years after their first description in animals. They too catalyse a myriad of reactions from the synthesis of plant products such as phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides and glucosinolates to the metabolism of herbicides. Plant P450s, often catalyse reactions which radically alter the chemical and biological activity of the substrate. For example, they define the functional activities of flavonoid natural products and the phytotoxic activity of herbicides. Plant P450s are therefore enzymes of major interest to natural product and crop protection chemists. However, there are significant difficulties in studying these enzymes. 1. They are encoded by large gene families and their functions cannot be predicted from their gene sequence. 2. The P450s are difficult to assay, isolate and purify, so classical biochemical methods are often ineffectual in identifying enzymes of interest. 3. These proteins are membrane bound and often difficult to express as a functional enzyme in recombinant host cells. Therefore, plant P450s represent a valuable target to apply novel technologies to functionally characterise family members.As a result of these difficulties, current research emphasis lies with the identification of P450 proteins cloned from cDNA sequences. This research area is advancing rapidly, however generating plant P450s in this way isn't without its problems. Most cloning approaches to plant P450s are hampered by the large sequence diversity of plant P450 genes and as such makes this technique difficult, therefore the direct approach to protein identification is the best way to access P450 proteins. The aim of this research fellowship is to synthesise chemical probes capable of being utilised with plant cells to directly and selectively identify P450 proteins. These probes will then enable the protein to be extracted and its sequence identified. Once identified, the corresponding P450 will be cloned and over-expressed in genetically engineered plant cells and its function determined.