Fig 1.
Oleic acid oxidation by GsCYP630B18 in combination with GsCPR1 or GsCPR2.
Extracted ion chromatograms (297.2–297.4 m/z, run in negative-ion ESI-MS) for oleic acid conversion by GsCYP630B18 in combination with GsCPR1 or GsCPR2 with the highest peak (m/z 297.3) identified as 18-hydroxyoleic acid. Darkened lines indicate where the peaks were integrated for relative ion intensity comparison. Samples GsCYP630 with GsCPR1 (top left panel) and GsCYP630 with GsCPR2 (top right panel) are shown in red. Controls (empty E. coli membrane fractions with GsCPR1or GsCPR2) are shown in black. Significant differences for the major product peak between controls and samples were indicated by P-value (P < 0.05, n = 3). Additional data for each analysis are shown below the LC/MS traces as m/z value, retention time, fold change as ratio of mean intensities between samples and controls, P-value, and peak intensity as average feature intensity within sample/control class.
Fig 2.
Single extracted ion chromatograms of RS1 and RS2 products.
Extracted ion chromatograms (297.2–297.6 m/z run in negative-ion ESI) from LC/MS analyses for GsCYP630B18 activity in RS1 and RS2 with oleic acid as substrate. Empty E. coli membrane fractions were used as control. Mass spectra representing hydroxyoleic acid peaks with RT 15.04 and 14.99 min are shown on the right.
Table 1.
Enzyme activity of GsCPR1 and GsCPR2.
Fig 3.
Standard plots for determining ferricyanide reduction kinetics catalyzed by GsCPR1 or GsCPR2 in the presence of NADPH.
The CPR-catalyzed reduction of ferricyanide in concentrations between 2 μM and 500 μM at 420 nm in the presence of saturating 100 mM NADPH in 100 mM potassium phosphate (pH 7.6). Different concentrations of ferricyanide are represented in shades of grey and dashed lines. Decreasing absorbance at 420 nm, quantifying the reduction of ferricyanide to ferrocyanide, was plotted by normalizing all points relative to the point zero baseline.
Table 2.
Calculated kinetic parameters for GsCPR1 and GsCPR2.
Fig 4.
Schematic representation of selective conservation of the CYP630-CPR2 gene cluster in Pezizomycotina.
The presence of the gene cluster found in seven Pezizomycotina classes is given as a fraction of the species that the gene cluster was identified in relative to all the species of a given class whose genomes were searched. Homologs of either gene were not identified outside of Pezizomycotina. The relative orientation of both genes is given (< >—divergent; > <—convergent, << or >>—co-oriented) [47]. The phylogeny was modeled after [48].
Fig 5.
Proposed model for the role of GsCYP630B18 in fatty acid oxidation.
The putative involvement of GsCYP630B18 is indicated by grey arrows. Triglycerides from the host plant in the lipoprotein are broken down to free fatty acids (FFA) and glycerol by the action of the lipoprotein lipase (LpL) [57]. Glycerol is further phosphorylated by glycerol kinase (GK) [58, 59] and enters glycolysis. On the cytosolic site, FFAs are activated and coupled to coenzyme A (CoA) by the catalysis of long-chain fatty acyl-CoA synthetases (ACSLs) [60] or by different fatty acid transporter proteins (FATPs) [61]. The transfer through the plasma membrane occurs by a protein-mediated mechanism. In the cell, FFAs can act at different sub-cellular localizations and have functions in energy generation and storage, membrane synthesis, protein modification, and activation of nuclear transcription factors [62]. Oxidation of FFA in fungi occurs mainly through β-oxidation in the mitochondrial matrix or peroxisomes, or through the ω-oxidation pathway in the endoplasmic reticulum. Several acyl-CoA ligases (ACLs) [63], involved in fatty acid metabolism, then attach CoA to the ends of fatty acids to form fatty-acyl-CoA. Fatty-acyl-CoA can pass through the outer mitochondrial membrane, but requires carnitine acetyl transferase (CT) [64] to cross the inner membrane. The multifunctional β-oxidation enzyme FOX2[65, 66] was induced in Gs mycelia grown on fatty acids, indicating possible peroxisomal oxidation. In ω-oxidation, the hydroxylase reaction is catalyzed by CYP630 (red bubble) and its redox partner CPR 2. Dicarboxylic acids are then subject to further β-oxidation. Gene IDs and respective changes in transcript abundance in Gs grown on fatty acids are given in Table 3 [14, 67].
Table 3.
Changes in transcript abundance of genes involved in the GsCYP630B18 putative fatty acid oxidation pathway following growth on monoterpenes, triglycerides or oleic acid as the sole carbon sources.
Fig 6.
The model of RS2 reconstitution system of the FMN domain (red) of GsCPR2 and GsCYP630B18 (orange), its substrate oleic acid (red) and the non-substrate stearic acid (yellow).
(A) The FMN-binding domain of GsCPR2 in open conformation interacts with the GsCYP630B18 that brings the FMN cofactor in close proximity to the CYP heme cofactor and thus facilitates electron transfer. The GsCYP630B18 transmembrane region is inserted into the lipid bilayer. (B) Docking of oleic acid (red) versus the stearic acid (yellow) in the active site of GsCYP630B18. Arginine 224 (green) holds both compounds in the active site channel.
Fig 7.
Time course of the root mean square (RMS) changes of the heme moiety in GsCYP630B18 with oleic acid (left) and stearic acid (right) as a substrate.
The protein was constrained for the first 100 ps.
Fig 8.
Amino acid alignment of 4 FMN-binding domains of GsCPR2 and GsCPR1, which interact with GsCYP630B18.
Loops are underlined and in bold. Lys 322 in loop 4 is marked in red.