Engineered Alkane-Hydroxylating Cytochrome P450BM3 Exhibiting Nativelike Catalytic Properties

Department of Chemistry and Chemical Engineering, California Institute of Technology, 1200 California Blvd. MC 210-41, Pasadena, CA 91125, USA.
Angewandte Chemie International Edition (Impact Factor: 11.26). 02/2007; 46(44):8414-8. DOI: 10.1002/anie.200702616
Source: PubMed


(Figure Presented) Divide, evolve, and conquer: A domain-based strategy (see scheme) was used to engineer high catalytic and coupling efficiency for propane hydroxylation in a multidomain cytochrome P450 enzyme. The engineered enzymes exhibit high total activities in whole-cell bioconversions of propane to propanol under mild conditions, using air as oxidant.


Available from: Rudi Fasan
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    • "Six mutants of the uncharged Ser72 and Ala74 residues that contained a negatively charged amino acid at these respective positions were selected. The Ser72 and Ala74 residues are located around the substrate binding channel (Li et al., 2001b; Otey et al., 2006; Dietrich et al., 2009) of the protein and have been shown to influence regioselectivity (Otey et al., 2006; Dietrich et al., 2009) and metabolic efficiency (Li et al., 2000, 2001b; Fasan et al., 2007) in previous studies. Two M01 mutants that contained mutations at the Ser72 position, MT43 (S72D) and MT44 (S72E), and one M01 mutant that contained a mutation at the Ala74 position (MT41; A74E) were selected. "
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    • "It is difficult to accurately measure the "in-cell conversion rate", but apparently the AMO/CPR-oxidation capability needs to be improved to fully convert the amorphadiene to artemisinic acid. Improvement of the kinetic properties of P450s is likely to be challenging, though such improvements have been demonstrated in a microbial system [28]. Nonetheless, genes/enzymes in plant secondary metabolism are evolutionarily recent, and hence there may be room for further improvement of their catalytic efficiencies by in vitro evolution or site-directed mutagenesis. "
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