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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 MdLOX1a mutants 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) Arg268Ala mutant. (b) Gly567Ala mutant. (c) Ile578Leu mutant. (d) Val582Phe mutant. 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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fig6: HPLC analysis of hydroxy fatty acids formed by MdLOX1a mutants 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) Arg268Ala mutant. (b) Gly567Ala mutant. (c) Ile578Leu mutant. (d) Val582Phe mutant. 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: Of these seven analyzed single mutants only Arg268Ala, Gly567Ala, Ile578Leu and Val582Phe showed high substrate conversion rates of LA, LnA and AA (60%–100% of LOX1:Md:1a activity with LA). The Thr775Leu mutant exhibited significant lower activity (∼33% of LOX1:Md:1a activity with LA), whereas LOX activity of the Ile566Phe and Leu572Ile mutants was nearly completely abolished. Hydroperoxy products formed by the different mutant LOX proteins showed regio- and stereospecificity similar to LOX1:Md:1a, except for Gly567Ala (Figure 6). The mutation of Gly567 to alanine affected an amino acid residue, which has been previously described to determine LOX stereoselectivity.47 Instead of forming a mixture of 13(R)- and 9(S)-HpODE, the Gly567Ala mutant protein acted as a strict 9-LOX producing mainly 9(S)-HpODE (about 87.5% of total hydroperoxides) from linoleic acid (Table 2). Also, reactions of the mutant enzyme with LnA and AA yielded product profiles similar to LOX1:Md:1c (Supplementary Table S4). Mutation of Val582 to the more space-filling phenylalanine did not change regio- or stereospecificity of the enzyme (Figure 6). The Val582Phe mutant still formed 13(R)-HpODE as the major product from LA. However, the low amounts of the 9-HpODE product consisted of a racemic mixture of both enantiomers (Table 2). Product profiles detected for the reaction with LnA and AA were similar to those observed for wild-type LOX1:Md:1a protein. In contrast, the major products of the Ile578Leu mutant with AA were 9-HpETE (29%) and 5-HpETE (21%) (Supplementary Table S4). Although the Ile578Leu mutant mainly formed 13-hydroperoxides from LA and LnA, a slight loss of regioselectivity was observed, leading to increased levels of the 9-hydroperoxides.


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 MdLOX1a mutants 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) Arg268Ala mutant. (b) Gly567Ala mutant. (c) Ile578Leu mutant. (d) Val582Phe mutant. 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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Related In: Results  -  Collection

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fig6: HPLC analysis of hydroxy fatty acids formed by MdLOX1a mutants 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) Arg268Ala mutant. (b) Gly567Ala mutant. (c) Ile578Leu mutant. (d) Val582Phe mutant. 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: Of these seven analyzed single mutants only Arg268Ala, Gly567Ala, Ile578Leu and Val582Phe showed high substrate conversion rates of LA, LnA and AA (60%–100% of LOX1:Md:1a activity with LA). The Thr775Leu mutant exhibited significant lower activity (∼33% of LOX1:Md:1a activity with LA), whereas LOX activity of the Ile566Phe and Leu572Ile mutants was nearly completely abolished. Hydroperoxy products formed by the different mutant LOX proteins showed regio- and stereospecificity similar to LOX1:Md:1a, except for Gly567Ala (Figure 6). The mutation of Gly567 to alanine affected an amino acid residue, which has been previously described to determine LOX stereoselectivity.47 Instead of forming a mixture of 13(R)- and 9(S)-HpODE, the Gly567Ala mutant protein acted as a strict 9-LOX producing mainly 9(S)-HpODE (about 87.5% of total hydroperoxides) from linoleic acid (Table 2). Also, reactions of the mutant enzyme with LnA and AA yielded product profiles similar to LOX1:Md:1c (Supplementary Table S4). Mutation of Val582 to the more space-filling phenylalanine did not change regio- or stereospecificity of the enzyme (Figure 6). The Val582Phe mutant still formed 13(R)-HpODE as the major product from LA. However, the low amounts of the 9-HpODE product consisted of a racemic mixture of both enantiomers (Table 2). Product profiles detected for the reaction with LnA and AA were similar to those observed for wild-type LOX1:Md:1a protein. In contrast, the major products of the Ile578Leu mutant with AA were 9-HpETE (29%) and 5-HpETE (21%) (Supplementary Table S4). Although the Ile578Leu mutant mainly formed 13-hydroperoxides from LA and LnA, a slight loss of regioselectivity was observed, leading to increased levels of the 9-hydroperoxides.

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.