Genome-to-function characterization of novel fungal P450 monooxygenases oxidizing polycyclic aromatic hydrocarbons (PAHs)

Abstract

Fungi, particularly the white rot basidiomycetes, have an extraordinary capability to degrade and/or mineralize (to CO(2)) the recalcitrant fused-ring high molecular weight (4 aromatic-rings) polycyclic aromatic hydrocarbons (HMW PAHs). Despite over 30years of research demonstrating involvement of P450 monooxygenation reactions in fungal metabolism of HMW PAHs, specific P450 monooxygenases responsible for oxidation of these compounds are not yet known. Here we report the first comprehensive identification and functional characterization of P450 monooxygenases capable of oxidizing different ring-size PAHs in the model white rot fungus Phanerochaete chrysosporium using a successful genome-to-function strategy. In a genome-wide P450 microarray screen, we identified six PAH-responsive P450 genes (Pc-pah1-Pc-pah6) inducible by PAHs of varying ring size, namely naphthalene, phenanthrene, pyrene, and benzo(a)pyrene (BaP). Using a co-expression strategy, cDNAs of the six Pc-Pah P450s were cloned and expressed in Pichia pastoris in conjunction with the homologous P450 oxidoreductase (Pc-POR). Each of the six recombinant P450 monooxygenases showed PAH-oxidizing activity albeit with varying substrate specificity towards PAHs (3-5 rings). All six P450s oxidized pyrene (4-ring) into two monohydroxylated products. Pc-Pah1 and Pc-Pah3 oxidized BaP (5-ring) to 3-hydroxyBaP whereas Pc-Pah4 and Pc-Pah6 oxidized phenanthrene (3-ring) to 3-, 4-, and 9-phenanthrol. These PAH-oxidizing P450s (493-547 aa) are structurally diverse and novel considering their low overall homology (12-23%) to mammalian counterparts. To our knowledge, this is the first report on specific fungal P450 monooxygenases with catalytic activity toward environmentally persistent and highly toxic HMW PAHs.

Figure 1

Effect of P450 inhibitor piperonyl butoxide (PB) on biodegradation of pyrene and benzo(a)pyrene by P. chrysosporium. Nutrient-rich malt extract broth cultures and negative controls were prepared as described under Materials and Methods section. Final concentrations of the test PAH and PB in the cultures were 20 ppm and 0.5 mM, respectively. Asterisk indicates statistically significant (P ≤0.05) effect of the inhibitor. The plotted values represent means ± standard deviations for three biological replicates. Abbreviations: Pyr, pyrene; BaP, benzo(a)pyrene; no-inh, no inhibitor.

Figure 2

Co-expression and activity characterization of Pc-Pah P450 monooxygenases and the homologous P450 reductase partner (Pc-POR) in Pichia pastoris. The expressed enzymes were assessed in the yeast microsomes prepared as described under Materials and Methods section. (A) SDS-PAGE analysis of the microsomal protein preparations from recombinant P450-POR yeast clones Pc-Pah1 through Pc-Pah6 (lanes 1–6) and negative control yeast (lane c). Proteins were separated on 7% SDS-PAGE and detected by staining with SYPRO Ruby (BioRad). The protein band coinciding with Pc-POR (estimated 81.6 kDa) is shown by an arrow. (B) Western blot analysis of Pc-Pah proteins using anti-his antibody. (C) Characteristic CO-difference spectra for the individual Pc-Pah proteins (Pc-Pah1 through Pc-Pah6) and negative control. (D) P450 enzyme concentrations and POR activity levels of the individual Pc-Pah clones versus the negative control (C). P450 concentration values are based on the CO-difference spectra shown in panel C. POR activity values are the means for three biological replicates (the SD values varied between 0.07 and 0.23).

Figure 3

Relative PAH-oxidizing activities of the six recombinant Pc-Pah P450 enzymes towards varying ring-size PAH compounds in whole cell assays. Each culture was spiked with a test PAH and the oxidation activity was assessed at 36 h of incubation. The values represent means ± standard deviations for three biological replicates and are statistically significant (P ≤0.05).

Figure 4

PAH oxygenation reactions catalyzed by the individual recombinant Pc-Pah enzymes (Pc-Pah1 to Pc-Pah6) of P. chrysosporium.