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A dual positional specific lipoxygenase functions in the generation of flavor compounds during climacteric ripening of apple.

Schiller D, Contreras C, Vogt J, Dunemann F, Defilippi BG, Beaudry R, Schwab W - Hortic Res (2015)

Bottom Line: Site-directed mutagenesis of Gly567 to an alanine converted the dual positional specific LOX1:Md:1a to an enzyme with a high specificity for 9(S)-hydroperoxide formation.The high expression level of the corresponding MdLOX1a gene in stored apple fruit, the genetic association with a quantitative trait locus for fruit ester and the remarkable agreement in regio- and stereoselectivity of the LOX1:Md:1a reaction with the overall LOX activity found in mature apple fruits, suggest a major physiological function of LOX1:Md:1a during climacteric ripening of apples.While LOX1:Md:1c, LOX2:Md:2a and LOX2:Md:2b may contribute to aldehyde production in immature fruit upon cell disruption our results furnish additional evidence that LOX1:Md:1a probably regulates the availability of precursors for ester production in intact fruit tissue.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology of Natural Products, Technische Universität München , Liesel-Beckmann-Str. 1, D-85354 Freising, Germany.

ABSTRACT
Lipoxygenase (LOX) is an important contributor to the formation of aroma-active C6 aldehydes in apple (Malus × domestica) fruit upon tissue disruption but little is known about its role in autonomously produced aroma volatiles from intact tissue. We explored the expression of 22 putative LOX genes in apple throughout ripening, but only six LOXs were expressed in a ripening-dependent manner. Recombinant LOX1:Md:1a, LOX1:Md:1c, LOX2:Md:2a and LOX2:Md:2b proteins showed 13/9-LOX, 9-LOX, 13/9-LOX and 13-LOX activity with linoleic acid, respectively. While products of LOX1:Md:1c and LOX2:Md:2b were S-configured, LOX1:Md:1a and LOX2:Md:2a formed 13(R)-hydroperoxides as major products. Site-directed mutagenesis of Gly567 to an alanine converted the dual positional specific LOX1:Md:1a to an enzyme with a high specificity for 9(S)-hydroperoxide formation. The high expression level of the corresponding MdLOX1a gene in stored apple fruit, the genetic association with a quantitative trait locus for fruit ester and the remarkable agreement in regio- and stereoselectivity of the LOX1:Md:1a reaction with the overall LOX activity found in mature apple fruits, suggest a major physiological function of LOX1:Md:1a during climacteric ripening of apples. While LOX1:Md:1c, LOX2:Md:2a and LOX2:Md:2b may contribute to aldehyde production in immature fruit upon cell disruption our results furnish additional evidence that LOX1:Md:1a probably regulates the availability of precursors for ester production in intact fruit tissue.

No MeSH data available.


HPLC analysis of hydroxy fatty acids formed by wild-type apple LOX enzymes followed by chemical reduction. Product profiles for LOX activity with linoleic acid were obtained via SP-HPLC. Ratios of (R)- and (S)-enantiomers were determined by CP-HPLC (Insets). (a) Wild-type LOX1:Md:1a. (b) Wild-type LOX1:Md:1c. (c) Wild-type LOX2:Md:2a. (d) Wild-type LOX2:Md:2b. A LiChrospher® 100 Diol column (125×4 mm, 5 µm) was eluted at 2 mL min−1 with hexane:2-propanol:formic acid (100:2:0.1, by vol.) with UV detection at 234 nm. Chiral phase HPLC analysis of enantiomer composition was conducted on a Chiralpak® IA column (250×4.6 mm, 5 µm) with an elution at 0.75 mL min−1 in hexane/ethanol/acetic acid (95:5:0.02, by vol.) and UV detection at 234 nm.
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fig4: HPLC analysis of hydroxy fatty acids formed by wild-type apple LOX enzymes followed by chemical reduction. Product profiles for LOX activity with linoleic acid were obtained via SP-HPLC. Ratios of (R)- and (S)-enantiomers were determined by CP-HPLC (Insets). (a) Wild-type LOX1:Md:1a. (b) Wild-type LOX1:Md:1c. (c) Wild-type LOX2:Md:2a. (d) Wild-type LOX2:Md:2b. A LiChrospher® 100 Diol column (125×4 mm, 5 µm) was eluted at 2 mL min−1 with hexane:2-propanol:formic acid (100:2:0.1, by vol.) with UV detection at 234 nm. Chiral phase HPLC analysis of enantiomer composition was conducted on a Chiralpak® IA column (250×4.6 mm, 5 µm) with an elution at 0.75 mL min−1 in hexane/ethanol/acetic acid (95:5:0.02, by vol.) and UV detection at 234 nm.

Mentions: Reaction products of LA, LnA and arachidonic acid (AA) formed by the four proteins were analyzed to characterize regio- and stereospecificity of the apple LOXs. Partially purified LOX1:Md:1a, LOX1:Md:1c, LOX2:Md:2a and LOX2:Md:2b proteins converted all three tested substrates to their corresponding hydroperoxy fatty acids, as was confirmed by LC-MS analysis (Supplementary Figure S6). Additionally, reaction products with LA were reduced to their corresponding hydroxy fatty acids and analyzed using SP-HPLC (Figure 4). Fractions of 9- and 13-HODE were collected and analyzed for enantiomer composition by CP-HPLC (Figure 4 insets). Both, LOX1:Md:1a and LOX1:Md:1c produced 9(S)-HpODE from linoleic acid (Figure 4a and 4b), but LOX1:Md:1a formed primarily 13(R)-HpODE (about 77% of total hydroperoxides). LOX1:Md:1a acted as a dual positional specific enzyme producing 13- and 9-HpODE in a ratio of 8:1 (Table 1). In contrast, 9(S)-HpODE was the major product of LOX1:Md:1c activity with LA, as only trace amounts of the regioisomer (0.5% of total hydroperoxides) and enantiomer (2.3%) was formed. Similar results were obtained for LOX activity of both enzymes with LnA (Supplementary Table S4). LOX2:Md:2a and LOX2:Md:2b converted LA and LnA predominantly to 13-hydroperoxides (Figure 4c and 4d). However, the main product of LOX2:Md:2b reaction with LA was identified as 13(S)-HpODE, whereas LOX2:Md:2a formed 13(R)- and 9(S)-HpODE in a ratio comparable to LOX1:Md:1a. Accordingly, LOX1:Md:1a and LOX2:Md:2a were classified as (R)-LOX with a 13(R)/9(S)-specificity. LOX1:Md:1c and LOX2:Md:2b proved to be (S)-LOX enzymes and were classified as linoleate 9(S)- and 13(S)-LOX, respectively.


A dual positional specific lipoxygenase functions in the generation of flavor compounds during climacteric ripening of apple.

Schiller D, Contreras C, Vogt J, Dunemann F, Defilippi BG, Beaudry R, Schwab W - Hortic Res (2015)

HPLC analysis of hydroxy fatty acids formed by wild-type apple LOX enzymes followed by chemical reduction. Product profiles for LOX activity with linoleic acid were obtained via SP-HPLC. Ratios of (R)- and (S)-enantiomers were determined by CP-HPLC (Insets). (a) Wild-type LOX1:Md:1a. (b) Wild-type LOX1:Md:1c. (c) Wild-type LOX2:Md:2a. (d) Wild-type LOX2:Md:2b. A LiChrospher® 100 Diol column (125×4 mm, 5 µm) was eluted at 2 mL min−1 with hexane:2-propanol:formic acid (100:2:0.1, by vol.) with UV detection at 234 nm. Chiral phase HPLC analysis of enantiomer composition was conducted on a Chiralpak® IA column (250×4.6 mm, 5 µm) with an elution at 0.75 mL min−1 in hexane/ethanol/acetic acid (95:5:0.02, by vol.) and UV detection at 234 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4595979&req=5

fig4: HPLC analysis of hydroxy fatty acids formed by wild-type apple LOX enzymes followed by chemical reduction. Product profiles for LOX activity with linoleic acid were obtained via SP-HPLC. Ratios of (R)- and (S)-enantiomers were determined by CP-HPLC (Insets). (a) Wild-type LOX1:Md:1a. (b) Wild-type LOX1:Md:1c. (c) Wild-type LOX2:Md:2a. (d) Wild-type LOX2:Md:2b. A LiChrospher® 100 Diol column (125×4 mm, 5 µm) was eluted at 2 mL min−1 with hexane:2-propanol:formic acid (100:2:0.1, by vol.) with UV detection at 234 nm. Chiral phase HPLC analysis of enantiomer composition was conducted on a Chiralpak® IA column (250×4.6 mm, 5 µm) with an elution at 0.75 mL min−1 in hexane/ethanol/acetic acid (95:5:0.02, by vol.) and UV detection at 234 nm.
Mentions: Reaction products of LA, LnA and arachidonic acid (AA) formed by the four proteins were analyzed to characterize regio- and stereospecificity of the apple LOXs. Partially purified LOX1:Md:1a, LOX1:Md:1c, LOX2:Md:2a and LOX2:Md:2b proteins converted all three tested substrates to their corresponding hydroperoxy fatty acids, as was confirmed by LC-MS analysis (Supplementary Figure S6). Additionally, reaction products with LA were reduced to their corresponding hydroxy fatty acids and analyzed using SP-HPLC (Figure 4). Fractions of 9- and 13-HODE were collected and analyzed for enantiomer composition by CP-HPLC (Figure 4 insets). Both, LOX1:Md:1a and LOX1:Md:1c produced 9(S)-HpODE from linoleic acid (Figure 4a and 4b), but LOX1:Md:1a formed primarily 13(R)-HpODE (about 77% of total hydroperoxides). LOX1:Md:1a acted as a dual positional specific enzyme producing 13- and 9-HpODE in a ratio of 8:1 (Table 1). In contrast, 9(S)-HpODE was the major product of LOX1:Md:1c activity with LA, as only trace amounts of the regioisomer (0.5% of total hydroperoxides) and enantiomer (2.3%) was formed. Similar results were obtained for LOX activity of both enzymes with LnA (Supplementary Table S4). LOX2:Md:2a and LOX2:Md:2b converted LA and LnA predominantly to 13-hydroperoxides (Figure 4c and 4d). However, the main product of LOX2:Md:2b reaction with LA was identified as 13(S)-HpODE, whereas LOX2:Md:2a formed 13(R)- and 9(S)-HpODE in a ratio comparable to LOX1:Md:1a. Accordingly, LOX1:Md:1a and LOX2:Md:2a were classified as (R)-LOX with a 13(R)/9(S)-specificity. LOX1:Md:1c and LOX2:Md:2b proved to be (S)-LOX enzymes and were classified as linoleate 9(S)- and 13(S)-LOX, respectively.

Bottom Line: Site-directed mutagenesis of Gly567 to an alanine converted the dual positional specific LOX1:Md:1a to an enzyme with a high specificity for 9(S)-hydroperoxide formation.The high expression level of the corresponding MdLOX1a gene in stored apple fruit, the genetic association with a quantitative trait locus for fruit ester and the remarkable agreement in regio- and stereoselectivity of the LOX1:Md:1a reaction with the overall LOX activity found in mature apple fruits, suggest a major physiological function of LOX1:Md:1a during climacteric ripening of apples.While LOX1:Md:1c, LOX2:Md:2a and LOX2:Md:2b may contribute to aldehyde production in immature fruit upon cell disruption our results furnish additional evidence that LOX1:Md:1a probably regulates the availability of precursors for ester production in intact fruit tissue.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology of Natural Products, Technische Universität München , Liesel-Beckmann-Str. 1, D-85354 Freising, Germany.

ABSTRACT
Lipoxygenase (LOX) is an important contributor to the formation of aroma-active C6 aldehydes in apple (Malus × domestica) fruit upon tissue disruption but little is known about its role in autonomously produced aroma volatiles from intact tissue. We explored the expression of 22 putative LOX genes in apple throughout ripening, but only six LOXs were expressed in a ripening-dependent manner. Recombinant LOX1:Md:1a, LOX1:Md:1c, LOX2:Md:2a and LOX2:Md:2b proteins showed 13/9-LOX, 9-LOX, 13/9-LOX and 13-LOX activity with linoleic acid, respectively. While products of LOX1:Md:1c and LOX2:Md:2b were S-configured, LOX1:Md:1a and LOX2:Md:2a formed 13(R)-hydroperoxides as major products. Site-directed mutagenesis of Gly567 to an alanine converted the dual positional specific LOX1:Md:1a to an enzyme with a high specificity for 9(S)-hydroperoxide formation. The high expression level of the corresponding MdLOX1a gene in stored apple fruit, the genetic association with a quantitative trait locus for fruit ester and the remarkable agreement in regio- and stereoselectivity of the LOX1:Md:1a reaction with the overall LOX activity found in mature apple fruits, suggest a major physiological function of LOX1:Md:1a during climacteric ripening of apples. While LOX1:Md:1c, LOX2:Md:2a and LOX2:Md:2b may contribute to aldehyde production in immature fruit upon cell disruption our results furnish additional evidence that LOX1:Md:1a probably regulates the availability of precursors for ester production in intact fruit tissue.

No MeSH data available.