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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.


Overview of the substrate binding pocket of LOX1:Md:1a. Homology model showing positions of amino-acid residues described to be essential for LOX activity and specificity (gray, blue) as well as positions chosen for site-directed mutagenesis (orange, blue). A 13(S)-HpODE molecule (green) illustrates position and depth of the substrate binding pocket. The bottom of the cavity is marked by Arg732, which is essential for enzyme activity and therefore highly conserved among plant LOX enzymes. However, the catalytic iron atom (red sphere) is coordinated by five conserved residues (His524, His529, His715, Asn719, Ile863) near the entrance. The residues Thr581 and Val582 align with the so-called Sloane position described for mammalian LOX and are known to control regiospecificity of fatty acid oxygenation. In addition, Gly567 has been described to influence both regio- and stereoselectivity of LOX enzymes.
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fig5: Overview of the substrate binding pocket of LOX1:Md:1a. Homology model showing positions of amino-acid residues described to be essential for LOX activity and specificity (gray, blue) as well as positions chosen for site-directed mutagenesis (orange, blue). A 13(S)-HpODE molecule (green) illustrates position and depth of the substrate binding pocket. The bottom of the cavity is marked by Arg732, which is essential for enzyme activity and therefore highly conserved among plant LOX enzymes. However, the catalytic iron atom (red sphere) is coordinated by five conserved residues (His524, His529, His715, Asn719, Ile863) near the entrance. The residues Thr581 and Val582 align with the so-called Sloane position described for mammalian LOX and are known to control regiospecificity of fatty acid oxygenation. In addition, Gly567 has been described to influence both regio- and stereoselectivity of LOX enzymes.

Mentions: To further evaluate the potential for the involvement of LOX1:Md:1a in autonomous aroma formation in ripe apple fruits, we investigated the determinants of LOX1:Md:1a specificity by site-directed mutagenesis of the MdLOX1a gene and subsequent biochemical analysis of the heterologously expressed mutant proteins. To compare our findings on enzymatic activity, we chose LOX1:Md:1c as a reference. Both apple LOXs share a sequence identity of 85% and a highly conserved active site with the putative 9-LOX-specific TV motif (Supplementary Figure S2, LOX1:Md:1a Thr581/Val582; LOX1:Md:1c Thr580/Val581) at the so-called Sloane position. However, only LOX1:Md:1c produced exclusively 9(S)-hydroperoxides from LA and LnA. Comparison of their primary structures revealed four positions that differed in the enzyme active sites of both proteins (Supplementary Figure S2). Thus, Gly567, Leu572, Ile578 and Thr775 in LOX1:Md:1a were individually replaced by the corresponding amino acids of LOX1:Md:1c, resulting in the single mutants Gly567Ala, Leu572Ile, Ile578Leu and Thr775Leu (Figure 5). Three additional positions were mutated to elucidate the role of an inverse head-to-tail orientation of the substrate in the active site on the production of 13-hydroperoxides by LOX1:Md:1a.46 In the straight substrate orientation, free fatty acids enter the hydrophobic cavity with their methyl end first. The carboxyl group is then supposed to form salt bridges with polar residues near the cavity entrance.50 In LOX1:Md:1a, the positions Arg268 and Lys283 were candidates for such a salt bridge formation. Their effect on product regiochemistry was analyzed by substitution of Arg268 with a hydrophobic alanine residue (Figure 5). In addition, Ile566 at the cavity entrance was substituted by the more space-filling phenylalanine residue of LOX1:Md:1c to analyze a potential steric hindrance of salt bridge formation. A similar effect should be observed for substitution of Val582 at the Sloane position by a phenylalanine residue. Arg268Ala and Ile566Phe should interfere with the interaction of the carboxyl group and polar residues at the cavity entrance, leading to a preference of inverse substrate orientation and thereby 9(S)-LOX activity, whereas Val582Phe should hinder salt bridge formation of the carboxyl group and the conserved arginine residue (Arg732) at the bottom of the pocket, resulting in a straight substrate orientation and 13(S)-LOX activity. In total, seven positions were chosen for site-directed mutagenesis (Figure 5).


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)

Overview of the substrate binding pocket of LOX1:Md:1a. Homology model showing positions of amino-acid residues described to be essential for LOX activity and specificity (gray, blue) as well as positions chosen for site-directed mutagenesis (orange, blue). A 13(S)-HpODE molecule (green) illustrates position and depth of the substrate binding pocket. The bottom of the cavity is marked by Arg732, which is essential for enzyme activity and therefore highly conserved among plant LOX enzymes. However, the catalytic iron atom (red sphere) is coordinated by five conserved residues (His524, His529, His715, Asn719, Ile863) near the entrance. The residues Thr581 and Val582 align with the so-called Sloane position described for mammalian LOX and are known to control regiospecificity of fatty acid oxygenation. In addition, Gly567 has been described to influence both regio- and stereoselectivity of LOX enzymes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4595979&req=5

fig5: Overview of the substrate binding pocket of LOX1:Md:1a. Homology model showing positions of amino-acid residues described to be essential for LOX activity and specificity (gray, blue) as well as positions chosen for site-directed mutagenesis (orange, blue). A 13(S)-HpODE molecule (green) illustrates position and depth of the substrate binding pocket. The bottom of the cavity is marked by Arg732, which is essential for enzyme activity and therefore highly conserved among plant LOX enzymes. However, the catalytic iron atom (red sphere) is coordinated by five conserved residues (His524, His529, His715, Asn719, Ile863) near the entrance. The residues Thr581 and Val582 align with the so-called Sloane position described for mammalian LOX and are known to control regiospecificity of fatty acid oxygenation. In addition, Gly567 has been described to influence both regio- and stereoselectivity of LOX enzymes.
Mentions: To further evaluate the potential for the involvement of LOX1:Md:1a in autonomous aroma formation in ripe apple fruits, we investigated the determinants of LOX1:Md:1a specificity by site-directed mutagenesis of the MdLOX1a gene and subsequent biochemical analysis of the heterologously expressed mutant proteins. To compare our findings on enzymatic activity, we chose LOX1:Md:1c as a reference. Both apple LOXs share a sequence identity of 85% and a highly conserved active site with the putative 9-LOX-specific TV motif (Supplementary Figure S2, LOX1:Md:1a Thr581/Val582; LOX1:Md:1c Thr580/Val581) at the so-called Sloane position. However, only LOX1:Md:1c produced exclusively 9(S)-hydroperoxides from LA and LnA. Comparison of their primary structures revealed four positions that differed in the enzyme active sites of both proteins (Supplementary Figure S2). Thus, Gly567, Leu572, Ile578 and Thr775 in LOX1:Md:1a were individually replaced by the corresponding amino acids of LOX1:Md:1c, resulting in the single mutants Gly567Ala, Leu572Ile, Ile578Leu and Thr775Leu (Figure 5). Three additional positions were mutated to elucidate the role of an inverse head-to-tail orientation of the substrate in the active site on the production of 13-hydroperoxides by LOX1:Md:1a.46 In the straight substrate orientation, free fatty acids enter the hydrophobic cavity with their methyl end first. The carboxyl group is then supposed to form salt bridges with polar residues near the cavity entrance.50 In LOX1:Md:1a, the positions Arg268 and Lys283 were candidates for such a salt bridge formation. Their effect on product regiochemistry was analyzed by substitution of Arg268 with a hydrophobic alanine residue (Figure 5). In addition, Ile566 at the cavity entrance was substituted by the more space-filling phenylalanine residue of LOX1:Md:1c to analyze a potential steric hindrance of salt bridge formation. A similar effect should be observed for substitution of Val582 at the Sloane position by a phenylalanine residue. Arg268Ala and Ile566Phe should interfere with the interaction of the carboxyl group and polar residues at the cavity entrance, leading to a preference of inverse substrate orientation and thereby 9(S)-LOX activity, whereas Val582Phe should hinder salt bridge formation of the carboxyl group and the conserved arginine residue (Arg732) at the bottom of the pocket, resulting in a straight substrate orientation and 13(S)-LOX activity. In total, seven positions were chosen for site-directed mutagenesis (Figure 5).

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.