Characterisation of cytochrome P450s ìn Anopheles gambiae

Cytochrome P450s provide a natural mechanism by which insects defend themselves against the relatively small number of insecticides approved by WHO in fighting malaria vectors. Three dimensional structures of these enzymes are important to aid our understanding of the mechanism of their action in dissipating the harmful effects of such insecticides. However, to date no coordinates of any insect P450 structure have been deposited in the Protein Data Base. In this study a C-terminally, His-tagged, recombinant CYP6Z2, CYP for cytochrome P450, was constructed genetically and its expression and biochemical properties evaluated in an attempt to produce material suitable for crystallisation trials and ultimately X-ray structural analysis.
The recombinant enzyme was purified in a soluble form through the introduction of a range of chemical agents including sodium cholate. A series of truncating mutants were constructed to determine the minimum set of primary structural elements that create a barrier to crystallisation. Genetically engineered truncations (11 amino acids and 22 amino acids) of the N-terminal hydrophobic region did not produce holoprotein. The N-terminal region appears to be essential to ensure the proper folding of insect P450 investigated, which may be the principal reason underpinning the lack of success in producing crystal structures for this group of enzymes.
The homology model of CYP6Z2 used in this thesis, compared with those of the confirmed pyrethroid metabolisers; CYP6D1 and CYP6P3, revealed three key amino acid differences at their active sites. Single and double mutations of these three amino acids were constructed to explore their individual and joint roles in the metabolic activities of CYP6Z2. The single mutants, CYP6Z2 (Y102F) and CYP6Z2 (F212L) retained similar affinities as wild type for both benzyloxyresorufin (BR) and methoxyresorufin (MR) fluorescent probes. However, the turnover number of CYP6Z2 (Y102F) was 1.7-fold lower than the wild type for BR but shows improved activity with MR achieving 2-fold increase in turnover. CYP6Z2 (F212L) maintained the same turnover number with the wild type against BR but attained a 3.9-fold increase in activity against the methoxy derivative. Deletion of the positively charged arginine in CYP6Z2 (R210A) significantly improved the activity of the enzyme. The turnover number increased by 3-fold against BR and the enzyme effectiveness also increased by 2-fold. To further investigate the roles of these residues, the same mutations were introduced into CYP6P3. The addition of a hydroxyl group in CYP6P3 (F110Y) changed the kinetic behaviour of the enzyme drastically: the mutant lost activity with the resorufin probe and the affinity for the DEF probe was lowered by 7-fold. Conversely, introduction of benzyl ring in CYP6P3 (L216F) did not affect the affinity, but the enzyme’s activity increased by 15-fold with DEF. CYP6P3 (L216F) also improved the pyrethroid metabolising ability of the enzyme. These experiments clearly point to the important contribution of these amino acids in modulating the metabolic characteristics of these P450s. Similar studies were carried out to investigate the function of F115 in CYP6Z2 and CYP6Z3. This residue is frequently found close to the heam in P450s, where it is believed to play a key stabilising role in substrate recognition. A range of mutants were prepared and all produced holoproteins except F115H. The results presented in this thesis, point to a significant role in metabolic function for this residue, F115A, in CYP6Z3, and a rationalisation of the data is presented.
Finally, the metabolic activities of CYP6Z2 and CYP6Z3 were compared in this study. Results presented here, suggest that both act independently in the presence of cytochrome b5 in their activities against the resorufin fluorescent probes but their activities with diethyl fluorescein (DEF) probe increased significantly when cytochrome b5 is present. CYP6Z3 has 2-fold turnover rate and 5-fold greater efficiency more than CYP6Z2 in dealkylation of BR and 8-fold and 10-fold in turnover and efficiency respectively for MR. These metabolic comparisons suggest strongly that CYP6Z3 is an improved “version” of CYP6Z2.