has been the most intensively studied model organism for understanding the physiology, biochemistry, and genetics of biodegradation of lignin and toxic chemical pollutants by basidiomycetous white rot fungi. Peroxidases and oxygenases including P450 monooxygenases have been shown to play important roles as initial oxidizing systems in this white rot fungus for biotransformation of different xenobiotics [1
]. Whole genome sequencing has revealed that this fungus carries a large P450 contingent comprising of about 150 P450 genes, arranged in 16 gene clusters that are grouped under existing 12 cytochrome P450 (CYP) families and 11 fungal CYP clans [3
], and a single P450 reductase component.
The NADPH-dependent cytochrome P450 oxidoreductase (POR, EC 220.127.116.11), formerly abbreviated as CPR, is known to serve as a common electron donor to multiple monooxygenases in a typical microsomal P450 system, although multiple PORs have been reported in certain plants and even fungi [5
]. The electron transfer proceeds from NADPH to the P450 heme via FAD and FMN domains of the POR. Typical type II microsomal eukaryotic P450 mono-oxygenases primarily obtain both the electrons, needed for their monooxygenation reaction, from the POR, although involvement of an alternate electron transfer mechanism via the cyt b5 reductase-cyt b5 chain in providing one of the two electrons (the second electron) from NADH to the P450 monooxygenase has been known [7
]. Cytochrome b5 reductase (EC 18.104.22.168, cyt b5r), a membrane-bound flavoprotein containing a single FAD as a prosthetic group, catalyzes the reduction of cytochrome b5 (cyt b5) utilizing NADH as an electron donor. Cytochrome b5 (cyt b5) is known to be involved in a number of oxidative reactions, which include metabolism of fatty acids, steroids, and endogenous compounds. The role of cyt b5 as an obligate partner and modifier in xenobiotic biotransformation has been documented for higher eukaryotes [7
], but such information is not available for filamentous fungi. Whereas cyt b5
and cyt b5r
are located on genome scaffold 4 (whole genome version 2.0), the POR
gene is located on scaffold 12 (whole genome version 2.0) of the P. chrysosporium
genome. None of the three genes co-localize with any of the known 16 P450 clusters in the P. chrysosporium
In addition to POR and the cyt b5r-cyt b5 complex, cytochrome P450 enzymes can also receive electrons from other protein partners in eukaryotes depending on their intracellular location and their physiological function. For example, type I mitochondrial P450 systems obtain electrons from adrenodoxin reductases (AdR). Specifically, AdRs receive electrons from NADPH and transfer them to the P450 enzymes via the [2Fe-2S]-ferredoxin-type carrier adrenodoxin [8
]. Type III P450s are self sufficient and do not require molecular oxygen or external electron source. Type IV proteins such as P450nor (nicA
) can transfer electrons directly from NADH/NADPH to its substrate nitric oxide [9
]. Type I, III, and IV P450 proteins have not been identified in the P450ome of P. chrysosporium
Considering the extraordinarily large nature of the P450 monooxygenase contingent (~150 P450 genes) in P. chrysosporium, the single P450 oxidoreductase (POR) enzyme present in this organism could be insufficient to cater to the electron transfer needs of all the P450 mono-oxygenases. Presently, little or no information is available with respect to gene regulation and function of the P450 redox proteins of the P450 enzyme system in P. chrysosporium. Therefore, characterization of these enzymes, including the primary redox protein POR and the alternate redox proteins cyt b5 and cyt b5 reductase, will facilitate understanding of the redox mechanisms during P450 monooxygenation reactions in this model biological system.