oleic acid

ChEBI · EMBL-EBI ↗ · Källdatum saknas · Källans villkor

An octadec-9-enoic acid in which the double bond at C-9 has Z (cis) stereochemistry.

NovakMetka; LahLjerka; ŠalaMartin; StojanJure; BohlmannJoerg; KomelRadovan; PorolloAlexey · Oleic Acid Metabolism via a Conserved Cytochrome P450 System-Mediated ω-Hydroxylation in the Bark Beetle-Associated Fungus Grosmannia clavigera ↗ · 20 mars 2015 · Källans villkor

The activity of GsCYP630B18 was tested in reconstitution systems with GsCPR1 and GsCPR2 (RS1 and RS2, respectively) and substrates as described previously [32]. For RS1 and RS2, a 0.2 ml membrane fraction of approximately 16.8 μM GsCYP630B18 and 0.2 ml of the yeast microsomes of 13 μM GsCPR1 and GsCPR2 for RS1 and RS2, respectively, was prepared in 50 mM Tris/HCl (pH 7.4)/0.1 mM EDTA/10% glycerol. The reactions were initiated by adding 0.1 M NADPH and substrate (50 μM oleic acid (C18:1), linoleic acid (C18:2), stearic acid (C18:0), palmitic acid (C16:0), palmitoleic acid (C16:1), myristic acid (C14:0), lauric acid (C12:0), capric acid (C10:0) or arachidonic acid (C20:4)). After one hour of incubation at RT the reactions were stopped. We extracted the organic product with methanol and analyzed it with LC/MS.

NovakMetka; LahLjerka; ŠalaMartin; StojanJure; BohlmannJoerg; KomelRadovan; PorolloAlexey · Oleic Acid Metabolism via a Conserved Cytochrome P450 System-Mediated ω-Hydroxylation in the Bark Beetle-Associated Fungus Grosmannia clavigera ↗ · 20 mars 2015 · Källans villkor

For functional characterization, we expressed GsCYP630B18 in two different reconstitution systems (RS), RS1 and RS2, with GsCPR1 and GsCPR2, respectively. We tested substrate conversion and specificity of RS1 or RS2 with saturated and unsaturated fatty acids of different chain lengths: oleic acid (C18:1), linoleic acid (C18:2), stearic acid (C18:0), palmitic acid (C16:0), palmitoleic acid (C16:1), myristic acid (C14:0), lauric acid (C12:0), capric acid (C10:0) and arachidonic acid (C20:4). Product formation was analyzed in methanol extracts by LC/MS. Both CPRs supported activity of GsCYP630B18 in in vitro assays, despite having different catalytic efficiencies. CYP630B18 converted oleic acid, but none of the other tested compounds (S1 and S2 Figs.).

NovakMetka; LahLjerka; ŠalaMartin; StojanJure; BohlmannJoerg; KomelRadovan; PorolloAlexey · Oleic Acid Metabolism via a Conserved Cytochrome P450 System-Mediated ω-Hydroxylation in the Bark Beetle-Associated Fungus Grosmannia clavigera ↗ · 20 mars 2015 · Källans villkor

The major peak of a single product of molecular weight 298 in the extracted ion chromatogram (Fig. 1) was identified by its fragmentation pattern as 18-hydroxyoleic acid which could be formed by either epoxidation or hydroxylation of oleic acid (S3 Fig., S4 Table). TOF/LC/MS confirmed the enzyme product as C18H33O3 (calculated for hydroxyoleic acid C18H33O3 - [M-H]-: 297.2508; found: 297.2435; difference 0.1ppm). Control microsomes without GsCYP630B18 did not oxidize any of the fatty acids (Fig. 2). Welch’s t-test showed highly significant differences between controls and samples for the presence of the major product peak. P-values for RS1 and RS2 were 0.0007 and 0.0057, respectively (Fig. 1).

NovakMetka; LahLjerka; ŠalaMartin; StojanJure; BohlmannJoerg; KomelRadovan; PorolloAlexey · Oleic Acid Metabolism via a Conserved Cytochrome P450 System-Mediated ω-Hydroxylation in the Bark Beetle-Associated Fungus Grosmannia clavigera ↗ · 20 mars 2015 · Källans villkor

The pattern of CYP630B-CPR2 gene expression in a closely related blue-stain fungus, Ophiostoma piceae, very much resembles the expression profiles in Gs, following the same treatment with triglycerides and oleic acid [20]. This comparison across species, which inhabit a similar micro-environment and occupy similar ecological niches, indicates that this CYP system could be an important alternative in fatty acid degradation. The ω-hydroxylation pathway is possibly a concurrent free fatty acid-removal mechanism when the major ß-oxidation pathways of fatty acids are ineffective or overwhelmed by high levels of potential substrates. Such would likely be the case when the fungus encounters saturating levels of toxic plant defense monoterpenes, triglycerides and free fatty acids, all of which it must process and channel into mitochondrial and peroxisomal ß-oxidation. We offer a pathway model for the role of GsCYP630B18 and fatty acid ω-hydroxylation in Gs during host colonization (Fig. 5). The high specificity of GsCYP630 for oleic acid hydroxylation and the considerable induction of the GsCYP630B18-CPR2 gene cluster following growth on triglycerides and oleic acid (Table 3), as well as its genomic organization suggest that this CYP system is an important detoxification mechanism for the removal of free oleic acid in this conifer-attacking fungus.

NovakMetka; LahLjerka; ŠalaMartin; StojanJure; BohlmannJoerg; KomelRadovan; PorolloAlexey · Oleic Acid Metabolism via a Conserved Cytochrome P450 System-Mediated ω-Hydroxylation in the Bark Beetle-Associated Fungus Grosmannia clavigera ↗ · 20 mars 2015 · Källans villkor

However, the biological roles of CPR paralogs in an organism remain unclear. The present work and earlier studies, report differences in gene expression levels of CPR under varying growth and treatment conditions, different catalytic properties and the fact that the choice of the native CPR redox partner affects CYP product specificity in in vitro experiments [16]. It was hypothesized that the CPR1 paralog was responsible for providing reducing equivalents to CYPs in endogenous primary metabolism, while the CPR2 possibly functions in specialized metabolism. Genomic localization and occurrence of the CPR2 paralog together with CYP630 in numerous Pezizomycotina genomes, their apparent joint gene expression profiles reported in Ophistomatales fungi grown on fatty acid substrates [14, 20], as well as functionality in oleic acid ω-hydroxylation via CYP630 indicate a narrower niche specialization of CPR2. However, this evidence does not rule out possible involvement of CPR2 as an alternative redox partner to other fungal CYPs found in a particular genome.