It is not surprising that the engineering of bacterial P450 systems has been focused on the best characterized systems, P450cam and P450BM-3. There have been significant successes from structure-based redesign, site saturation mutagenesis, and directed evolution by error-prone PCR as well as chimera construction [90-92]. All these methods have limitations but the results to date have been very promising. Furthermore, these approaches are increasingly being combined to further enhance activity and selectivity. P450cam and P450BM-3 have been engineered to oxidize large molecules such as pyrene, small molecules such as propane and ethane, highly branched fatty acids, as well as complex molecules such as terpenes, alicyclic compounds and pharmaceuticals.
In general, it is relatively straightforward to obtain fast NAD( P) H turnover rates but the coupling yield and especially selectivity of product formation are far more challenging. P450BM-3 in particular appears to be finely balanced between the slow reacting substrate-free form and fast electron transfer upon structural perturbations induced by mutations. For instance, the R47L/Y51F/F87A, A74G/ F87V/L188Q, 1-12G and 9-10A mutants all show fast NADPH turnover rates with a range of substrates. However, in many cases the reactions are uncoupled and non-selective. Refinement of screening methods (e.g. by the use of alcohol dehydrogenases that can discriminate between primary and secondary alcohols) will play an increasingly important role.
Recent directed evolution of the CYP102 family of enzymes has imparted terminal alkane oxidation activity to these enzymes which naturally target subterminal positions [66, 67]. Similarly P450cam has been engineered for fast limonene oxidation but perillyl alcohol, a desirable terpenoid that arises from oxidation of a methyl substituent, was not observed (Scheme 4.14). Instead products from oxidation at the more reactive olefinic double bond and allylic CH2 groups dominated . On the other hand the CYP153 family of enzymes are highly selective for terminal C-H bond oxidation, converting octane to 1-octanol and limonene to perillyl alcohol with almost total selectivity . Therefore the discovery of novel activity by isolation and characterization of new P450 enzymes from diverse organisms still has a crucial role to play, together with protein engineering, for developing P450 biooxidation applications.
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