Oleic Acid Metabolism <i>via</i> a Conserved Cytochrome P450 System-Mediated ω-Hydroxylation in the Bark Beetle-Associated Fungus <i>Grosmannia clavigera</i>

<div><p>The bark beetle-associated fungus <i>Grosmannia clavigera</i> participates in the large-scale destruction of pine forests. In the tree, it must tolerate saturating levels of toxic conifer defense chemicals (e.g. monoterpenes). The fungus can metabolize some of these compounds through the ß-oxidation pathway and use them as a source of carbon. It also uses carbon from pine triglycerides, where oleic acid is the most common fatty acid. High levels of free fatty acids, however, are toxic and can cause additional stress during host colonization. Fatty acids induce expression of neighboring genes encoding a cytochrome P450 (CYP630B18) and its redox partner, cytochrome P450 reductase (CPR2). The aim of this work was to study the function of this novel P450 system. Using LC/MS, we biochemically characterized CYP630 as a highly specific oleic acid ω-hydroxylase. We explain oleic acid specificity using protein interaction modeling. Our results underscore the importance of ω-oxidation when the main ß-oxidation pathway may be overwhelmed by other substrates such as host terpenoid compounds. Because this CYP-CPR gene cluster is evolutionarily conserved, our work has implications for metabolism studies in other fungi.</p></div

Schematic representation of selective conservation of the CYP630-CPR2 gene cluster in Pezizomycotina.

<p>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) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120119#pone.0120119.ref047" target="_blank">47</a>]. The phylogeny was modeled after [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120119#pone.0120119.ref048" target="_blank">48</a>].</p

Calculated kinetic parameters for <i>Gs</i>CPR1 and <i>Gs</i>CPR2.

<p>Calculated kinetic parameters for <i>Gs</i>CPR1 and <i>Gs</i>CPR2.</p

Time course of the root mean square (RMS) changes of the heme moiety in <i>Gs</i>CYP630B18 with oleic acid (left) and stearic acid (right) as a substrate.

<p>The protein was constrained for the first 100 ps.</p

Oleic acid oxidation by <i>Gs</i>CYP630B18 in combination with <i>Gs</i>CPR1 or <i>Gs</i>CPR2.

<p>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.</p

Standard plots for determining ferricyanide reduction kinetics catalyzed by <i>Gs</i>CPR1 or <i>Gs</i>CPR2 in the presence of NADPH.

<p>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.</p

Changes in transcript abundance of genes involved in the <i>Gs</i>CYP630B18 putative fatty acid oxidation pathway following growth on monoterpenes, triglycerides or oleic acid as the sole carbon sources.

<p>RNA-Seq data for selected genes from mycelia grown for 10 days on YNB minimal medium with a mixture of monoterpenes (YNB+MT) (monoterpenes: (+)-limonene, (+)-3-carene, racemic α-pinene and (−)-β-pinene at a ratio of 5:3:1:1), for 5 days with triglycerides (YNB+TG), or oleic acid (YNB+OA) as the sole carbon source, relative to controls grown on mannose [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120119#pone.0120119.ref014" target="_blank">14</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120119#pone.0120119.ref067" target="_blank">67</a>].</p><p>*<i>P-</i>values are not significant.</p><p>LpL—lipoprotein lipase, GK—glycerol kinase, ACSL—long-chain fatty acyl-CoA synthetase, FATPs—fatty acid transporter proteins, ACLs—acyl-CoA coenzymes, CT—carnitine acetyl transferase, FOX2—multifunctional β-oxidation enzyme, CYP630B18—cytochrome P450, CPR2—cytochrome P450 reductase 2.</p><p>Changes in transcript abundance of genes involved in the <i>Gs</i>CYP630B18 putative fatty acid oxidation pathway following growth on monoterpenes, triglycerides or oleic acid as the sole carbon sources.</p

Enzyme activity of <i>Gs</i>CPR1 and <i>Gs</i>CPR2.

<p>EU—one enzyme unit of CPR reduces 1.0 μmol oxidized cytochrome c per minute in the presence of 100 mM NADPH at pH 7.8 and 25°C.</p><p>m—membrane-bound</p><p>Positive control—rabbit liver CPR</p><p>Negative control—membrane fraction of yeast strain expressing empty plasmid pYEDP60U</p><p>Enzyme activity of <i>Gs</i>CPR1 and <i>Gs</i>CPR2.</p

K18 S230T may form an additional hydrogen bond within the K18 chain in the L12 linker.

<p>Molecular dynamics experiments were performed on the K8/K18 L12 linker model with the duration of 1 ns. This was repeated 4 times for the wild type sequence and 5 times for the mutant. The resulting data was used to calculate: (A) the average distances between the hydroxyl group of SER/THR230 and the backbone oxygen of ALA226 in K18, which in the case of the mutant (THR230) falls within the range of a moderately strong hydrogen bond (2.5–3.2 Å); (B) the relative root mean square (RMS) along the run, a measure of dimer stability. Both parameters indicate that the K18 S230T mutation may be forming an additional hydrogen bond within the K18 chain, which would be expected to increase the rigidity of this part of protein to additional conformational pressures.</p

K8/K18 mutants have altered growth rates.

<p>Cells were grown on 24-well cell culture plates. After a week in culture cells were trypsinized and counted. As shown, the K8 mutants grow at a much higher rate than the K8 WT cell line, while the K18 S230T cells grow very slow, having only a quarter of the growth rate of K18 WT cells. All cell lines (mutant and wild type) have a normal cell cycle profile (not shown).</p