Malonyl-coenzyme A:21-Hydroxypregnane 21-O-Malonyltransferase Isoforms in Digitalis lanata

Recent research on elucidating cardenolide biosynthesis focused on the formation of the butenolide ring characteristic for cardenolides. It was suggested that the butenolide ring is formed from appropriate pregnane-21-O-malonyl hemiesters without participation of an enzyme [1].

Malonyl-coenzymeA:21-hydroxypregnane-21-O-malonyltransferase (21MaT) is an enzyme that catalyzes the formation of pregnane-21-O-malonyl hemiesters by transferring a malonyl moiety from malonyl-coenzymeA onto suitable 21-hydroxypregnane derivatives. Even though 21MaT was characterized and purified from Digitalis purpurea [3], genes encoding 21MaTs have not yet been identified.

We here reinvestigated enzymatic pregnane 21-O-malonylation and found that more than one 21MaT isoform is present in D. lanata. 21MaTs convert a broader substrate range than previously assumed [2] and can be distinguished on the basis of their acyl-acceptor specificity. Fractionated ammonium sulfate precipitation separates two isoforms, one preferring 5β-21-hydroxypregnane derivatives and one preferring the corresponding 5α- and Δ⁴-derivatives. Subsequent chromatofocusing of the fractions indicated the existence of a third isoform. The different isoforms prefer either 3β-O-acetyl-5β-pregane-14β,21-diol-20-one (21MaT1; IEP app. pH 5.5), desoxycorticosterone (21MaT2, IEP app. pH 5.3) or 5β-preganane-3,20-dion-21-ol (21MaT3, IEP app. pH 5.0) as substrate. 21MaTs may hence have a role not only in cardenolide metabolism, which is also supported by demonstrating 21MaT activity in the cardenolide-free species Arabidopsis thaliana [3]. Different 21MaT isoforms are now further characterized and purified focusing on the purification of 21MaT2.

[1] Pádua, R. M, et al. Tetrahedron 2016, 72: 4556 – 4563.

[2] Kuate, S. P, et al. Phytochemistry 2008, 69: 619 – 626.

[3] Höhn, S. Dissertation, in preparation