The cytochrome P450 monooxygenases ("P450s") constitute a large superfamily of heme-thiolate proteins widely distributed in different life forms including prokaryotes (archaea, bacteria), lower eukaryotes (fungi, insects), and higher eukaryotes (plants and animals). P450s play an important role in the metabolism of a wide variety of endogenous and xenobiotic compounds. The current P450 nomenclature [1
] is based on divergent evolution of the P450 superfamily. On the basis of sequence homology, all P450s can be categorized into two main classes, B ('bacterial') and E ('eukaryotic') [2
]. Bacterial P450s with three component systems [an FAD-containing flavoprotein (NADPH or NADH-dependent reductase), an iron-sulphur protein, and the P450 hemeprotein] and the fungal P450nor (CYP55) [3
] belong to the 'B'-class. All the other known P450s from distinct systems, including eukaryotic P450s and bacterial P450s such as P450BM3 (CYP102) from Bacillus megaterium
], belong to the 'E'-class. The eukaryotic microsomal P450 system contains two components, the NADPH:P450 oxidoreductase (POR), a flavoprotein containing both FAD and FMN, and the P450 monooxygenase containing the heme domain. The prokaryotic (bacterial) soluble P450 monooxygenase P450BM3 (CYP102) exists as a single protein with both heme and flavin functional domains. Typically, the bacterial P450s are soluble and shorter (approximately 400 amino acids) in length, whereas the eukaryotic P450s average around 500–600 amino acids or larger and are membrane-bound. The amino acid (aa) sequence of these P450 monooxygenase proteins is extremely diverse, with levels of identity as low as 16% in some cases, but their structural fold has remained the same throughout evolution. The existing data suggest that divergence of the P450 superfamily into B and E classes, and further divergence into stable P450 groups within the E class, is very ancient and had occurred before the appearance of eukaryotes [5
]. From the phylogenetic classification point of view, families have been identified based primarily on amino acid sequence similarity, with less than 40% similarity defining a family and 40–55% similarity defining a sub-family. Recently, the concept of "Clan" which represents higher order grouping of P450 families is gaining wider acceptance in the P450 community [6
Among the different phyla, plants have the highest number of P450 sequences, followed by fungi. So far, more than 380 fungal P450s have been identified and the number is increasing with the sequencing of new fungal genomes http://drnelson.utmem.edu/cytochromeP450.html
. In comparison to the yeast forms (Saccharomyces and Candida), the higher order fungi have a larger number of P450 sequences in their genomes, with 41 P450 genes predicted in Neurospora crassa
, 111 in Aspergillus spp
., 123 in Magnaporthe grisea
, 110 in Fusarium graminearum
and about 150 in Phanerochaete chrysosporium.
Assuming the possibility that the present day P450s have evolved from a single ancestor P450 gene, the large disparity in the P450 gene count among the different phyla and genera is reflective of the differences in metabolic demand. According to Nelson [8
], while plants have invested heavily on biochemistry for development of survival strategies thereby driving the P450s to expand rapidly, animals have invested on development of higher order sensory systems and hence carry comparatively fewer P450s. Fungi, which resemble plants in their sedentary habitat, appear to have driven P450s to diversify rapidly.
, the model white rot fungus, has the extraordinary ability to degrade and mineralize (to CO2
) lignin, the earth's most abundant aromatic polymer, and a wide range of toxic chemical pollutants. Lignin biodegradation occurs under nutrient-limited conditions when the fungus enters secondary metabolism. Only white rot fungi are known to possess the ability to completely degrade lignin [9
]. The working hypothesis is that initial depolymerization of the lignin by extracellular peroxidases releases chemical compounds that are internalized and further degraded by diverse intracellular enzymes, including P450 monooxygenases. Further, several studies have shown that this fungus can degrade and mineralize a broad spectrum of aromatic, alicyclic, and aliphatic chemical pollutants under both nutrient-limited (ligninolytic) and nutrient-sufficient (non-ligninolytic) conditions. Lately, P450 monooxygenases have been shown to be involved in several of these biotransformations and this has led to an increasing interest in the characterization of cytochrome P450 systems in this model white rot fungus.
Initial cloning efforts from this laboratory identified the first complete P450 system genes in P. chrysosporium
. These efforts led to the isolation [12
] and functional characterization [14
] of three full-length P450 genes pc
-2 and pc
-3 that were assigned to CYP63 family, and the P450 oxidoreductase gene (POR
) that is responsible for electron transfer to the multiple P450 monooxygenases [16
]. Subsequent whole genome sequencing by the Joint Genome Institute (JGI) of the US Department of Energy (US-DOE) has led to the identification of a large P450 contingent (P450ome) in P. chrysosporium
]. The P450 genes constitute nearly 1% of the coding sequences in the genome of this basidiomycetous fungus [13
]. Of the nearly 150 P450 monooxygenase genes identified in P. chrysosporium
genome, 108 have been assembled full-length and tentatively annotated based on general overall sequence homology analysis [13
]. To date, this is the highest number of P450s identified in any fungus, which appears to be one of the major underlying factors for its extraordinary catalytic potential. Understanding the structural and evolutionary aspects of such large family of P450s in conjunction with their physiological characteristics will help understand the functional versatility of this fungus. This study reports a detailed genome-wide structural and phylogenetic analysis of the P450ome of P. chrysosporium
, coupled with cloning and characterization of new cDNAs for selected multigene P450 families, with a broader aim to facilitate the classification/nomenclature and to understand the functional diversity and evolution of the P450s in this wood-rotting model white rot fungus.