H2O2-dependent cytochrome P450s that can catalyze monooxygenation of nonnative substrates were constructed by one-point mutagenesis.

Shorter-alkyl-chain fatty acids such as tridecanoic acid or lauric acid were produced from myristic acid by CYP152 peroxygenases.

Highly effective dipeptidic decoy molecules, which stimulate the direct hydroxylation of benzene by wild-type cytochrome P450BM3, were successfully developed through a rationally designed screening method. Extensive synthesis and step-wise screening of over 600 dipeptide derivatives were performed for the efficient evolution of decoy molecules. In the presence of N-(3-cyclopentyl)propanoyl-L-pipecolyl-L-phenylalanine (3CPPA-Pip-Phe), one of the most effective decoy molecules discovered herein, the catalytic turnover frequency and total turnover number for benzene hydroxylation reached 405 min −1 P450BM3 −1 and 54,500 P450BM3 −1 , respectively. Furthermore, the decoy molecules developed in this work drastically accelerated the hydroxylation of other non-native substrates, such as anisole and toluene, as well as nonaromatic compounds, such as cyclohexane, propane, and ethane. Using Nenanthoyl-L-pipecolyl-L-phenylalanine (C7AM-Pip-Phe), the hydroxylation rate for ethane to ethanol reached 82.7 min −1 P450BM3 −1 .

Haem substitution is an effective approach to tweak the function of haemoproteins. Herein, we report a facile haem substitution method for self-sufficient cytochrome P450BM3 (CYP102A1) from Bacillus megaterium utilising the transpeptidase Sortase A from Staphylococcus aureus. We successfully constructed Mn-substituted BM3 and investigated its catalytic activity.