Functional Characterization of a Melon Alcohol Acyl-transferase Gene Family Involved in the Biosynthesis of Ester Volatiles. Identification of the Crucial Role of a Threonine Residue for Enzyme Activity*

Abstract

Volatile esters, a major class of compounds contributing to the aroma of many fruit, are synthesized by alcohol acyl-transferases (AAT). We demonstrate here that, in Charentais melon (Cucumis melo var. cantalupensis), AAT are encoded by a gene family of at least four members with amino acid identity ranging from 84% (Cm-AAT1/Cm-AAT2) and 58% (Cm-AAT1/Cm-AAT3) to only 22% (Cm-AAT1/Cm-AAT4). All encoded proteins, except Cm-AAT2, were enzymatically active upon expression in yeast and show differential substrate preferences. Cm-AAT1 protein produces a wide range of short and long-chain acyl esters but has strong preference for the formation of E-2-hexenyl acetate and hexyl hexanoate. Cm-AAT3 also accepts a wide range of substrates but with very strong preference for producing benzyl acetate. Cm-AAT4 is almost exclusively devoted to the formation of acetates, with strong preference for cinnamoyl acetate. Site directed mutagenesis demonstrated that the failure of Cm-AAT2 to produce volatile esters is related to the presence of a 268-alanine residue instead of threonine as in all active AAT proteins. Mutating 268-A into 268-T of Cm-AAT2 restored enzyme activity, while mutating 268-T into 268-A abolished activity of Cm-AAT1. Activities of all three proteins measured with the prefered substrates sharply increase during fruit ripening. The expression of all Cm-AAT genes is up-regulated during ripening and inhibited in antisense ACC oxidase melons and in fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. The data presented in this work suggest that the multiplicity of AAT genes accounts for the great diversity of esters formed in melon.

A comparative analysis of characteristic floral scent compounds in Prunus mume and related species

“…29,30) Quantitative analysis of the endogenous extracts and the headspace volatiles of different plant species indicated that only a small amount of compounds was emitted from the flower in 1 h and most of the compounds remained in the floral tissue. This observation suggests that the emission of stored volatile compounds from floral tissues may be controlled, perhaps by an excretory process regulated at the cellular level.…”

Biosynthesis of plant‐derived flavor compounds

“…Alcohol acyl transferases (AAT) are capable of combining various alcohols and acyl CoAs, resulting in the synthesis of a wide range of esters, thus accounting for the diversity of esters. Numerous AAT genes have been isolated and characterized in fruit and vegetables (Aharoni et al, 2000;Beekwilder et al, 2004;El-Sharkawy et al, 2005). Aliphatic esters contribute to the aroma of nearly all fruits and are emitted by vegetative tissues.…”

Biochemistry of apple aroma: A review

“…In ripe apples, alcohols constitute 6 to 16 % of the total volatiles (8), whereas esters represent, depending on the apple variety, from 80 % in Golden Delicious and Golden Reinders (14,45) to 98 % in Starking Delicious (36). Esters can have linear or branched chains, and their concentration depends on the availability of precursors (37,46,47) and on the selectivity and activity of the enzymes involved (48)(49)(50)(51). Butyl acetate, hexyl acetate, 2-methylbutyl acetate and ethyl 2-methyl--butanoate are the most important esters due to their high content and impact on apple aroma of several varieties (6,52).…”

“…The diversity of esters formed by AAT enzymes can be infl uenced by substrate availability, the specifi city of the enzyme, or by the variety of genes encoding ATT enzymes (49)(50)(51). Even though AAT has more affi nity for linear than for branched alcohols (17), because acetyl-CoA is the most abundant CoA in fruits, most esters are acetate esters (33,117).…”