Cytochrome P450BM3 (P450BM3) is one of the most promising P450s for construction of biocatalysts because of its high monooxygenase activity. We found that P450BM3 starts to catalyze hydroxylation of nonnative substrates in the presence of perfluorinated carboxylic acids (PFCs) as inert dummy substrates (decoy molecules). PFCs initiate the activation of molecular oxygen in the same manner as with long-alkyl-chain fatty acids and induce the generation of compound I, but the compound I hydroxylates gaseous alkanes and benzene because the C–F bonds of PFCs are not oxidizable (Fig. 1a). We have succeeded in developing the next generation of decoy molecules by modifying the carboxylate of PFCs with amino acids and succeeded in enhancing the catalytic activity for gaseous alkanes. Amongst the next generation of decoy molecules examined, N-perfluorononanoyl-L-leucine (PFC9-L-Leu) was the most effective for hydroxylation of propane (256/min/P450) and ethane (45/min/P450). The crystal structure analysis of PFC9-L-Trp-bound form of P450BM3 showed that the terminal of alkyl chain does not reach to the active site (Fig. 1b). More recently, we have demonstrated that various carboxylic acids modified with amino acids (N-acyl amino acids) as well as amino acid dimers having a completely different structure from fatty acids can serve as decoy molecules. Benzene was more efficiently hydroxylated in the presence of these decoy molecules.

Hydroxylation of gaseous alkanes and benzene catalyzed by cytochrome P450BM3 using decoy molecules as a substrate analogue

255th National Meeting (2018)

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