Abstract
The aroma of peach fruit is predominantly determined by the accumulation of γ-decalactone and ester compounds. A previous study showed that the biosynthesis of these aroma compounds in peach fruit is catalyzed by PpAAT1, an alcohol acyltransferase. In this work, we investigated the key active site residues responsible for γ-decalactone and ester biosynthesis. A total of 14 candidate amino acid residues possibly involved in internal esterification and 9 candidate amino acid residues possibly involved in esterification of PpAAT1 were assessed via site-directed mutagenesis. Analyses of the in vitro enzyme activities of PpAAT1 and its site-directed mutant proteins (PpAAT1-SMs) with different amino acid residue mutations as well as the contents of γ-decalactone in transgenic tobacco leaves and peach fruits transiently expressing PpAAT1 and PpAAT1-SMs revealed that site-directed mutation of H165 in the conserved HxxxD motif led to lost enzymatic activity of PpAAT1 in both internal esterification and its reactions, whereas mutation of the key amino acid residue D376 led to the total loss of γ-decalactone biosynthesis activity of PpAAT1. Mutations of 9 and 7 other amino acid residues also dramatically affected the enzymatic activity of PpAAT1 in the internal esterification and esterification reactions, respectively. Our findings provide a biochemical foundation for the mechanical biosynthesis of γ-decalactone and ester compounds catalyzed by PpAAT1 in peach fruits, which could be used to guide the molecular breeding of new peach species with more favorable aromas for consumers.
Highlights
As an olfactory component of flavors, fruit aroma is an important index for fruit quality evaluation[1,2,3,4]
To identify the amino acids participating in enzyme–substrate recognition in the esterification reaction, acetyl–CoA and the four alcohols were docked to the active center of PpAAT1 to form a binding conformation (Fig. 1b)
Based on computer modeling and molecular docking analyses, 16 candidate amino acid residues involved in the internal esterification reaction and 9 candidate amino acid residues involved in the esterification reaction of PpAAT1 were predicted (Fig. 1a, d–g)
Summary
As an olfactory component of flavors, fruit aroma is an important index for fruit quality evaluation[1,2,3,4]. The unique aroma of different fruits depends on their volatile profiles, which are mainly composed of esters, lactones, aldehydes, and alcohol terpenoids[5,6]. Motif of vinorine synthase, a member of the BAHD acyltransferase family catalyzing acetyl–CoA- or CoAdependent vinorine formation, led to the loss of its enzymatic activity[19]. Similar results were observed with the substitution of H166 and D170 in the HxxxD motif of alcohol acyltransferase (VpAAT1), which catalyzes ester biosynthesis in mountain papaya[7,20]. PpAAT1, like other known AATs that contain an HxxxD-type acyltransferase-like motif (HAMCD), could catalyze both the internal esterification reaction at the hydroxyl (–OH) and –CoA groups of 4hydroxydecanoyl–CoA and the esterification reaction between alcohols and acyl-CoAs14
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
