In silico, in vitro, and structural investigations on BAHD-enzymes from different species able to malonylate 21-hydroxypregnanes

BAHD-acyltransferases represent a large enzyme family found ubiquitously in angiosperms. They are considered substrate-promiscuous, accepting various substrates ranging from small aliphatic alcohols to complex structures such as terpenoids or flavonoids. BAHDs catalyze the formation of esters using different acyl-CoA thioesters and are thus important enzymes of specialized plant metabolism [1]. It is assumed that one step in cardenolide formation, the 21-O-malonylation of 21-hydroxypregnanes, is catalyzed by a BAHD-malonyltransferase (21MaT).

To screen potential candidates able to malonate 21-hydroxypregnanes, we isolated cDNAs of BAHD-malonyltransferases from Arabidopsis thaliana (AtPMaT1, AtPMaT2), Erysimum crepidifolium (EcMaT1) and Digitalis lanata (DlMaT1). To predict substrate acceptance, homology models of these enzymes were generated. Docking simulations with those models implied that all tested enzymes, except AtPMaT2, accept 21-hydroxypregnanes as substrates [2].

To verify the in silico results, the candidate genes were expressed in E. coli and the respective enzymes tested for 21MaT activity in vitro. All enzymes accepted 21-hydroxypregnanes as substrates. However, AtPMaT1 turned out to have a remarkably higher specific 21MaT activity (20 – 700 times higher) as compared to the other enzymes tested.

To investigate this phenomenom, homology models of the various enzymes were compared.

AtPMaT1 and EcMaT1 show a high sequence identity but show considerable differences in enzyme activity in vitro. A comparison of the binding pockets revealed that their sequences only differ in two amino acids. To enlighten the catalytic role of these residues, site-directed mutagenesis experiments were performed and revealed that mutants of AtPMaT1 show an up to 100-fold decrease in 21MaT activity.

Publikationsverlauf

Artikel online veröffentlicht:
13. Dezember 2021

• References

• 1 Kruse LH, Weige AT, Martínez-Gómez J, Chobirko JD, Schaffer JE, Bennett A, Specht CD, Jez JM, Shukla D, Moghe GD. Ancestral class-promiscuity as a driver of functional diversity in the BAHD acyltransferase family in plants. bioRvix 2020 https://doi.org/10.1101/2020.11.18.385815.
• 2 Tropper M, Höhn S, Wolf L-S, Fritsch J, Kastner-Detter N, Rieck C, Munkert J, Meitinger N, Lanig H, Kreis W. 21-Hydroxypregnane 21-O-malonylation, a crucial step in cardenolide biosynthesis, can be achieved by substrate-promiscuous BAHD-type phenolic glucoside malonyltransferases from Arabidopsis thaliana and homolog proteins from Digitalis lanata. Phytochemistry 2021 https://doi.org/10.1016/j.phytochem.2021.112710