Degradation of lipoxygenase-derived oxylipins by glyoxysomes from sunflower and cucumber cotyledons

Abstract

Background: Oilseed germination is characterized by the degradation of storage lipids. It may proceed either via the direct action of a triacylglycerol lipase, or in certain plant species via a specific lipid body 13-lipoxygenase. For the involvement of a lipoxygenase previous results suggested that the hydroxy- or oxo-group that is being introduced into the fatty acid backbone by this lipoxygenase forms a barrier to continuous β-oxidation.

Results: This study shows however that a complete degradation of oxygenated fatty acids is possible by isolated cucumber and sunflower glyoxysomes. Interestingly, degradation is accompanied by the formation of saturated short chain acyl-CoAs with chain length between 4 and 12 carbon atoms lacking the hydroxy- or oxo-diene system of the oxygenated fatty acid substrate. The presence of these CoA esters suggests the involvement of a specific reduction of the diene system at a chain length of 12 carbon atoms including conversion of the hydroxy-group at C7.

Conclusions: To our knowledge this metabolic pathway has not been described for the degradation of polyunsaturated fatty acids so far. It may represent a new principle to degrade oxygenated fatty acid derivatives formed by lipoxygenases or chemical oxidation initiated by reactive oxygen species.

Figure 1

Turnover of LA and 13-HOD by glyoxysomes from etiolated cucumber (A/B) or sunflower (C/D) in the optical β-oxidation assay. The figures show one typical experiment while the mean values for all experiments are given in Table 1. A/C: Initial rates for NADH-formation from different fatty acid concentrations were fitted according to a hyperbolic equation. Comparable rates of NADH-formation and S_0.5 values for turnover of 13-HOD by glyoxysomes from cucumber (A) or sunflower (C) are observed, but only weak rates of NADH-formation from LA by sunflower organelles can be detected. B/D: The final concentrations of NADH were calculated from the final absorbance at 340 nm and the formed molecules NADH from each molecule of fatty acid was obtained from the slope of a linear fit. Either all data points or only the concentrations up to 5 μM were used for the fitting procedure. The latter value is less influenced by inactivation effects and is thought to give a more precise result. Again, comparable values for turnover of 13-HOD are obtained for both plants, but NADH formation from LA is strongly decreased when sunflower glyoxysomes are used.

Figure 2

Possible pathways for degradation of 13-HOD-CoA. Three rounds of classical β-oxidation reactions yield 7-hydroxy-12:2-CoA as the last intermediate which still contains the initial hydroxy-diene system. The first round of β-oxidation is exemplarily depicted with all expected intermediates. Note that in absence of NAD⁺ the conversion of 13-HOD (or LA) stalls at the 3-hydroxy-intermediate (see Figure 3). Intermediates that have been detected by HPLC or ESI-MS/MS are framed.

Figure 3

Time course of activation of LA by glyoxysomes from etiolated cucumber (A) or sunflower (B). β-oxidation assays were prepared as described in the material and method section with the only exception, that NAD⁺ was excluded from the assay. A: Activation of LA by glyoxysomes from cucumber. LA is directly activated to LA-CoA and converted to the 3-hydroxylated form. Small amounts of 13-HOD and 13-HOD-CoA are also observed. B: Activation of LA by glyoxysomes from sunflower. LA is rapidly oxidized to 13-HOD which is subsequently activated to 13-HOD-CoA and converted to 3-hydroxy-HOD-CoA. Small amounts of LA-CoA are also detected. HPLC-traces are shown at 234 nm, absorption signals of the individual intermediates are indicated by open (234 nm, conjugated double bond) or closed (260 nm, CoA-ester) circles.

Figure 4

Time course of turnover of 13-HOD by glyoxysomes from etiolated cucumber. β-oxidation assays were prepared as described in the material and method section. HPLC-traces after different reaction times were detected at 260 nm (A) and 234 nm (B). Absorption signals of the individual intermediates are indicated by open (234 nm, conjugated double bond) or closed (260 nm, CoA-ester) circles and open squares (280 nm, keto-diene system). Besides 13-HOD-CoA (retention time ~ 100 min) acyl-CoAs bearing the conjugated hydroxy-diene system after one (retention time ~ 96 min) and two (retention time ~ 92 min) cycles of β-oxidation are detected and have been validated by MS. The intermediate appearing after 120 min of reaction (retention time ~ 89 min, absorption at 234 and 260 nm) is very likely the 13-HOD-intermediate after three cycles of β-oxidation. Intermediates lacking the 234 nm absorption coelute with authentic acyl-CoA standards with chain length between 4 and 12 carbons. Intermediates lacking the 260 nm absorbtion are presumed to be 13-HOD after one (retention time ~ 111 min) or two (retention time ~ 100 min) rounds of β-oxidation.

Figure 5

Time course of turnover of 13-HOD by glyoxysomes from etiolated sunflower. β-oxidation assays were prepared as described in the material and method section. HPLC-traces after different reaction times were detected at 260 nm (A) and 234 nm (B). Absorption signals of the individual intermediates are indicated by open (234 nm, conjugated double bond) or closed (260 nm, CoA-ester) circles and open squares (280 nm, keto-diene system). Besides 13-HOD-CoA (retention time ~ 101 min) acyl-CoAs bearing the conjugated hydroxy-diene system after one (retention time ~ 97 min) and two (retention time ~ 93 min) cycles of β-oxidation are detected and have been validated by MS. The respective oxidized KOD-derivatives elute with slightly elongated retention times (~ 102, 98 and 94 min). Intermediates lacking the 234 nm absorption coelute with authentic acyl-CoA standards with chain length between 4 and 12 carbons.