Abstract
Cytochrome P450 monooxygenases are versatile heme-thiolate enzymes that catalyze a wide range of reactions. Self-sufficient cytochrome P450 enzymes contain the redox partners in a single polypeptide chain. Here, we present the crystal structure of full-length CYP116B46, a self-sufficient P450. The continuous polypeptide chain comprises three functional domains, which align well with the direction of electrons traveling from FMN to the heme through the [2Fe-2S] cluster. FMN and the [2Fe-2S] cluster are positioned closely, which facilitates efficient electron shuttling. The edge-to-edge straight-line distance between the [2Fe-2S] cluster and heme is approx. 25.3 Å. The role of several residues located between the [2Fe-2S] cluster and heme in the catalytic reaction is probed in mutagenesis experiments. These findings not only provide insights into the intramolecular electron transfer of self-sufficient P450s, but are also of interest for biotechnological applications of self-sufficient P450s.
Highlights
Cytochrome P450 monooxygenases are versatile heme-thiolate enzymes that catalyze a wide range of reactions
Self-sufficient P450 enzymes that contain the redox partners in a single polypeptide chain are attractive biocatalysts for many biotechnological applications, as there is no need to search for suitable redox partners to establish a functional system
An N-terminal P450 heme domain is fused to a C-terminal NADPH:cytochrome P450 reductase (CPR) that contains a flavin mononucleotide (FMN)-binding flavodoxin and a FAD/NADPHbinding domain (Supplementary Fig. 1a)
Summary
Cytochrome P450 monooxygenases are versatile heme-thiolate enzymes that catalyze a wide range of reactions. Self-sufficient cytochrome P450 enzymes contain the redox partners in a single polypeptide chain. It is suggested that P450BM3 functions as a homodimer[16], which would enable a close approach of cofactors and efficient electron transfer among redox centers from either autologous or neighboring domain[17,18]. In this regard, P450BM3 may employ both intramolecular and intermolecular interactions to transport electrons. The path for the electrons to travel from the redox partners to the heme group within the polypeptide chain of a self-sufficient P450 remains unclear due to the lack of structural information of the full-length protein. In order to investigate the molecular mechanism of electron transfer of selfsufficient P450, we have solved and report the crystal structure of full-length CYP116B46 from Tepidiphilus thermophilus[21]
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