Fig. 1. Simplified catalytic cycle of natural heme-dependent oxygenases.

Fig. 2. Active sites of UPO from) Agrocybe aegerita (PDB ID: 2YP1) (A and Marasmius rotula (PDB ID: 5FUJ) (B). The hydrogen bonds and measured distances (?) are drawn by dashed lines in yellow and black, respectively. The acid-base pairs and heme are shown as stick models in white and magenta, respectively.

Fig. 3. Calcitriol and typical 25-hydroxy-Grundmann's ketone generated by PaDa-I.

Fig. 4. Enantioenrichment of representative organoperoxides by PaDa-I.

Fig. 5. Halogenation and oxygenation modes of PaDa-I-H are fine-tuned by pH values.

Fig. 6. Peroxygenation of N-heterocycles using the lyophilized PaDa-I-H.

Fig. 7. Representative reactions catalyzed by PaDa-I derived mutants.

Fig. 8. Structural alignment of PaDa-I (PDB ID: 5OXU, colored in white) and Fett (PDB ID: 7PN6, colored in yellow). The heme cavity is narrowed by introducing a bulky leucine residue. The cavities are shown as surface models.

Fig. 9. Representative reactions catalyzed by engineered short UPOs.

Fig. 10. Active sites of P450SPα in complex with Palmitic acid (PDB ID: 3AWM) (A) and (R)-ibuprofen (PDB ID: 3VM4) (B). The hydrogen bonds and measured distances (?) are drawn by dashed lines in yellow and black, respectively. The substrate, decoy molecule, and heme are shown as stick models in green, yellow and magenta, respectively.

Fig. 11. Styrene oxidation catalyzed by P450SPα and P450BSβ with the assistance of decoy molecules.

Fig. 12. Representative reactions of catalytic selectivity modulated by P450BSβ mutants.

Fig. 13. Active sites of P450BSβ mutants in complex with Palmitic acid (PDB ID: 7WYG) (A) and Palmitoleic acid (PDB ID: 8HKD) (B). The hydrogen bonds and measured distances (?) are drawn by dashed lines in yellow and black, respectively. The substrate and heme are shown as stick models in green and magenta, respectively.

Fig. 14. Active site of CYP199A4 in complex with 4-methoxybenzoic acid (PDB ID: 7REH). The hydrogen bonds and measured distances (?) are drawn by dashed lines in yellow and black, respectively. The substrate and heme are shown as stick models in green and magenta, respectively.

Fig. 15. (A) The conceptual graph of DFSM-facilitated P450 peroxygenase. The DFSM is fixed at the specific site of P450 enzyme by the anchoring group, while the catalytic group is responsible for H2O2 activation. (B) The chemical structure of Im-C6-Phe. The anchoring and catalytic groups are labelled as green rectangle and magenta circle, respectively.

Fig. 16. The catalytic performance of (R)-enantioselective epoxidation of styrene and its derivatives.

Fig. 17. Typical demethylation reactions catalysed by P450BM3 mutants with the assistance of Im-C6-Phe/Im-C5-Phe.

Fig. 18. Typical hydroxylation reactions of alkanes (C3?C6) catalysed by P450BM3 mutants with the assistance of Im-C6-Phe.

Fig. 19. Representative long-term hydroxylation reactions of alkylbenzenes catalysed by P450BM3 with the addition of Im-C5-Phe.

Fig. 20. Active site of the P450BM3 mutants in complex with DFSMs. (A) Toluene (PDB ID: 7YDB); (B) ethylbenzene (PDB ID: 7YD9); (C) propylbenzene (PDB ID: 7YDD); (D) indane (PDB ID: 7YFT). DFSMs, heme and alkylbenzenes are shown as stick models in yellow, magenta, and cyan, respectively.

Fig. 21. The evolution of novel DFSMs and their catalytic performances. (A) The idea of evolving dipeptide based novel artificial cofactor. (B) Hydrogen-bonding networks and hydrophobic interactions between the enzyme and Im-C6-Phe-Phe. The hydrophobic binding pocket is denoted as stick and surface models colored in white. The hydrogen bonds and measured distances are shown by dashed lines in yellow and black, respectively. (C) The chemical structures of novel DFSMs. Each DFSM contains a catalytic base (imidazole, amine or pyridine) and a dipeptide-liked anchoring group (R1, R2: side chain of natural or unnatural amino acid). Binding affinity and reaction performance of novel DFSMs. (D) Binding affinity of representative novel DFSMs. (E) Concentration gradient experiments of representative DFSMs for styrene epoxidation reaction.