Digitalis lanata, L.

Tropper, Marina, H, Stephanie, ohn, Wolf, Laura-Sophie, Fritsch, Julia, Kastner-Detter, Nina, Rieck, Christoph, Munkert, Jennifer, Meitinger, Nadine, Lanig, Harald & Kreis, Wolfgang, 2021, 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 (112710) 187, pp. 1-10 : 6

2.4. BAHD-type malonyltransferase genes from Digitalis lanata

Focusing once again on the role of 21- O -malonylation in cardenolide formation in Digitalis , we performed a function-based search for BAHD-type malonyltransferases in the transcriptome database of D. purpurea (https://medicinalplantgenomics.msu.edu). This search resulted in 9 hits. The respective sequences were annotated as either putative malonyl-CoA:anthocyanin 5- O -glucoside-6 ′′ - O -malonyltransferases or putative quercetin 3- O -glucoside 6 ′′ - O -malonyltransferases. The sequences were checked for completeness by protein BLASTs using the NCBI databases ( Altschul et al., 1990). Finally, two promising candidates were identified, both annotated as quercetin 3- O -glucoside-6 ′′ - O -malonyltransferases, encoding for proteins with molecular masses of 47 kDa and 50 kDa, respectively.

The first sequence isolated from D. lanata , termed Dlmat1 (GenBank MT992078 ), contained 1374 nucleotides coding for 457 amino acids and had a molecular mass of 51.0 kDa. Dlmat1 was 94% identical to a sequence in the D. purpurea genome annotated as quercetin 3- O -glucoside-6 ′′ - O -malonyltransferase. BLAST searches also revealed high sequence identities with BAHD-type malonyltransferases from Dorcoceras hygrometricum Bunge and Sesamum indicum L. It was confirmed that the four motifs conserved among all members of the BAHD enzyme superfamily ( D’ Auria, 2006; Tuominen et al., 2011) are also present in Dl MaT1. The clade-Ia-specific motif LTFFD ( Tuominen et al., 2011) as well as the anthocyanin metabolism related motif NYFGNC ( Yu et al., 2009) were also identified. Based on this isolated sequence we were able to isolate three further genes coding for putative BAHD-type malonyltransferases (Dlmat2, GenBank MW013542 ; Dlmat3, GenBank MW013543 ; Dlmat4, GenBank MW013544 ), sharing nucleotide sequence identities ranging from 49.6% to 92.1% and amino acid sequence identities from 32.1% to 93.4% with each other and Atpmat1. The protein sequences were further analyzed using SignalP ( Nielsen, 2017) and DeepLoc-1.0 ( Almagro Armenteros et al., 2017). All of them were predicted to be ‘soluble’ proteins as already described for several BAHD-type enzymes ( D’ Auria 2006; Bontpart et al., 2015).