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 .

Biological methane oxidation is a highly desirable method for the conversion of natural gas into a liquid to meet the increasing demand for fuel and chemical feedstock as well as reducing the potent greenhouse effects of methane emissions. Because natural hemoenzymes that can catalyze the conversion of methane to methanol have not been found, it has long been considered that hemoenzymes, including cytochrome P450s (P450s), cannot catalyze the oxidative conversion of methane. Herein, we report the catalytic oxidation of methane by wild-type P450BM3, without any mutagenesis, in the presence of chemically evolved dummy substrates (decoy molecules) under high-pressure methane at 10 MPa. Our studies showed that methane was catalytically converted into methanol at room temperature with a total turnover number of 4.

Abbildung 1. a) Struktur des aktiven Zentrums von P450BM3 mit gebundenem N-Palmitoylglyzin (PDB ID: 1JPZ) oder b) C7-Pro-Phe (PDB ID: 6K58). c) Plausibler Mechanismus der Benzolhydroxylierung von P450BM3 mit C7-Pro-Phe als Täuschmolekül. d) Konzept dieser Studie, worin die Ganzzellbiotransformation Benzols zu Phenolen von rekombinantem E. coli, das P450BM3 exprimiert, katalysiert wird. Dies wird von einem gentechnisch veränderten Außenmembranprotein, das die Zellaufnahme von Täuschmolekülen und Benzol unterstützt, gefördert.

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TRICKING CYTOCHROME P450BM3: CATALYSIS OF VARIOUS NON-NATIVE SUBSTRATE TRANSFORMATIONS USING DECOY MOLECULES. In order to accomplish a greener chemistry, enzymes, such as the fatty-acid hydroxylase cytochrome P450BM3, have garnered increasing attention as potential candidates for the development of potent biocatalysts in recent years. However, one of the biggest issues hampering the quick and efficient application of P450BM3 as a biocatalyst lies in its stringent substrate specificity. Consequently, diverse mutagenesis-based approaches have been successfully employed as a means to alter the substrate specificity of P450BM3, leading to the generation of a myriad of highly specialised mutant variants. Nevertheless, repeated exhaustive mutagenesis is a laborious process with no guarantee for success, thus, alternative methods to more easily alter the enzyme’s substrate specificity have become increasingly desirable. In recent years, decoy molecules, which possess the ability to deceive wild-type P450BM3 into hydroxylating a range of non-native substrates, have emerged as such a “simpler” alternative. Within this review, focus will be placed upon the process underlying the development of these decoy molecules, which will be discussed in great detail. Furthermore, a summary of recent developments pertaining to the potential applications of decoy molecules from the development of a whole-cell biocatalyst to their use in crystallography will be discussed.