Pathway analysis of branched-chain ester biosynthesis in apple using deuterium labeling and enantioselective gas chromatography-mass spectrometry

The biosynthesis of volatile esters by Red Delicious apples was investigated by incubating fruit tissue with deuterated flavor precursors at various times after controlled atmosphere (CA) storage and measuring deuterium incorporation into branched-chain ester volatiles. 2-Methylbutyl acetate was the only volatile not significantly reduced by CA storage. Conversion of 2-methylbutanol to 2-methylbutyl acetate and of 2-methylbutanoic acid to ethyl 2-methylbutanoate and to hexyl 2-methylbutanoate was limited by the availability of 2-methylbutyl substrates but not by acetyl-CoA, ethanol, or hexanol, respectively. The enzymatic activity required for these reactions declined during CA storage. The conversion of 2-methylbutanoic acid to 2-methylbutanol was also substrate limited, but enzymic activity appeared stable in storage. Biosynthesis of both 2-methylbutanoic acid and 2-methylbutanol, from isoleucine, was severely depressed under CA storage. The reduced metabolism of isoleucine to 2-methylbutanoyl-CoA may be the primary reason for reduced branched-chain ester synthesis in CA-stored Red Delicious apples. Enantioselective gas chromatography-mass spectrometry confirmed that the chirality of (S)-2-methylbutyl acetate derives from L-isoleucine with the other enzymes in this pathway not being enantiospecific. Treatment of tissue samples with 2-methylbut-2E-enal gave only (S)-2-methylbutyl acetate, indicating that biosynthesis was not via tiglyl-CoA.