Cytochromes P450nor and P450foxy of the fungus Fusarium oxysporum

Abstract

Fusarium oxysporum contains two self-sufficient P450 species, P450nor (CYP55) and P450foxy (CYP505), both of which can complete their catalyses without the aid of other proteinaceous components and receive electrons directly from NADH or NADPH. P450nor reduces nitric oxide to nitrous oxide and is essential for fungal denitrification. Its tertiary structure is fundamentally the same as those of other P450s, whereas characteristic features, the presence of a big space and a positive charge cluster in the heme-distal pocket, correspond to the unique catalytic mechanism of P450nor. We studied the reaction mechanism of P450nor by analyzing various mutant proteins and by carrying out X-ray crystallography to obtain the following conclusion: (1) The distal positive charge cluster plays a crucial role in attracting and binding NADH. (2) The specificity against NADH and NADPH is mainly determined by the B′-helix. (3) The anion hole near the heme creates an electrophilic environment for the electrons to be transferred from NADH to the heme. P450foxy catalyzes ω-1∼ ω-3 hydroxylation of fatty acids. Cloning and expression in the heterologous yeast cells of its gene showed that it is a fused protein of P450 and its reductase. It was also shown that not only the P450 domain, but also the reductase domain of P450foxy, exhibits high amino acid sequence identities to the respective domains of P450BM3 of Bacillus megaterium. As F. oxysporum and B. megaterium belong to eukaryote and prokaryote, respectively, occurrence of the fused proteins with high similarity across phyla evokes interest with respect to the evolution of the P450 superfamily.