Thermostabilization of a cytochrome p450 peroxygenase.

Cytochrome P450 BM-3 is a soluble fatty acid hydroxylase composed of a heme domain and a reductase domain on a single polypeptide chain. We recently described a laboratoryevolved variant of the P450 BM-3 heme domain which functions as an H2O2-driven hydroxylase (TMperoxygenase∫) and does not require NADPH, O2, or the reductase. This variant, which we named 21B3, allows us to carry out cytochrome P450-catalyzed biotransformations under highly simplified reaction conditions: only the heme domain and hydrogen peroxide are needed for substrate (fatty acid) hydroxylation. Because its heme domain alone is competent for catalysis, P450 BM-3 peroxygenase 21B3 offers a unique opportunity to create a thermostable, functional cytochrome P450. Here we report further directed evolution of the 21B3 peroxygenase, resulting in an enzyme which is significantly more thermostable than wildtype cytochrome P450 BM-3 and retains much of the peroxygenase activity of 21B3. Enzymes are often poorly stable under conditions encountered during production, storage, or use. Improving enzyme resistance to thermal denaturation has been a major focus of protein engineering efforts. Improved thermostability often correlates with longer shelf-life, longer life-time during use (even at low temperatures), and a higher temperature optimum for activity. 7] There have been no reports of stabilizing the relatively unstable cytochrome P450 enzymes by protein engineering, however, primarily because the P450s comprise multiple subunits and contain thermolabile cofactors. P450 BM-3 heme domain containing the single amino acid substitution F87A (mutant HF87A) is significantly more active than wildtype heme domain (HWT) in reactions driven by H2O2. 9] Variant 21B3 is much more active than HF87A in 10 mM H2O2, but is also less thermostable than HWT and HF87A. We therefore sought to improve the thermostability of 21B3 while maintaining its improved peroxygenase activity. Six cycles of random mutagenesis or DNA shuffling and screening for retention of peroxygenase activity after heat treatment (see the Methods section) yielded the thermostable peroxygenase variant 5H6. To characterize the thermostability of the peroxygenase variants identified during screening, we measured the fraction of folded heme domain remaining after heat-treatment, which we determined from the fraction of the ferrous heme-CO complex that retained the 450 nm absorbance peak characteristic of properly-folded P450. Figure 1 shows the percentage of