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. "Spontaneous butenolide ring formation of pregnane-21-O-malonyl hemiesters under mild reaction conditions is facilitated by the 14β-hydroxy group present in all natural cardenolides." Tetrahedron 72.30 (2016): 4556-4563.
[2] Kuate, S. P., et al. "Purification and characterization of malonyl-coenzyme A: 21-hydroxypregnane 21-O-malonyltransferase (Dp21MaT) from leaves of Digitalis purpurea L." Phytochemistry 69.3 (2008): 619-626.
[3] Höhn, S. Dissertation, in preparation