5 research outputs found
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
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
Biochemical and Catalytic Properties of Three Recombinant Alcohol Acyltransferases of Melon. Sulfur-Containing Ester Formation, Regulatory Role of CoA-SH in Activity, and Sequence Elements Conferring Substrate Preference
Alcohol acyltransferases (AAT) play a key role in the biosynthesis of ester aroma volatiles in fruit. Three ripening-specific recombinant AATs of cantaloupe Charentais melon fruit (Cm-AAT1, Cm-AAT3, and Cm-AAT4) are capable of synthesizing thioether esters with Cm-AAT1 being by far the most active. All proteins, as well as AAT(s) extracted from melon fruit, are active as tetramers of around 200 kDa. Kinetic analysis demonstrated that CoA-SH, a product of the reaction, is an activator at low concentrations and an inhibitor at higher concentrations. This was confirmed by the addition of phosphotransacetylase at various concentrations, capable of modulating the level of CoA-SH in the reaction medium. Site-directed mutagenesis of some amino acids that were specific to the Cm- AAT sequences into amino acids that were consensus to other characterized AATs greatly affected the selectivity of the original protein and the number of esters produced