This document outlines the putative biosynthetic pathway of cardiac glycosides in Digitalis purpurea. The pathway involves three main stages: terpenoid backbone biosynthesis, steroid biosynthesis, and cardenolide biosynthesis. The terpenoid backbone biosynthesis involves the mevalonate (MVA) pathway and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The steroid biosynthesis involves enzymes that convert acetyl-CoA to various sterols. The cardenolide biosynthesis stage involves enzymes that modify sterols to produce cardenolide compounds through hydroxylation, acetylation, and glycosylation reactions. Key enzymes are identified for each stage of the pathway.

1. OHH
O
OP
OP
O
OH
OH
D-Glyceraldehyde
3-phosphate
1-Deoxy-D-xylulose
5-phosphate
2-C-Methyl-D-eryth
ritol 4-phosphate
OP
OH OH
OH
4-(Cytidine 5'-diphospho)
-2-C-methyl-D-erythritol
O
HO OH
OH
P
O
OH
O
N
N
HO OH
O
NH2
P O
O
OH
2-Phospho-4-(cytidine 5'-diphospho)
-2-C-methyl-D-erythritol
O
OH OH
OP
P
O
OH
O
N
N
HO OH
O
NH2
P O
O
OH
2-C-Methyl-D-erythritol
2,4-cyclodiphosphate
O
OH OH
O P
O
O
OH
P
HO O
1-Hydroxy-2-methyl-
2-butenyl 4-diphosphate
O
OH
PP
3-Hydroxy-3-methylglutaryl-CoA
HO S-CoA
OHO O
Mevalonic acid
HO OH
OHO
(R)-5-Phosphomevalonate
HO O
OHO
PP
(R)-5-Diphosphomevalonate
HO O
OHO
PP
Isopentenyl diphosphate
O
CH2
PP
Presqualene diphosphate
O PP
H
H
Squalene
(S)-Squalene-2,3-epoxide
O
Cycloartenol
HO
H
H
H
24-Methylenecycloartanol
HO
H
H
H
CH2
Cycloeucalenol
HO
H
H
H
CH2
Obtusifoliol
HO
H
H
CH2
¦Ä8,14 -Sterol
HO
H
H
CH2
4¦Á -Methylfecosterol
HO
H
H
H
CH2H
24-Methylene lophenol
HO
CH2
Episterol
HO
CH2
5-Dehydroepisterol
HO
CH2
24-Methylenecholesterol
HO
CH2
¦Ä 7-Avenasterol
HO
HC
5-Dehydroavenasterol
HO
HC
Sitosterol
HO
H
Stigmasterol
HO
CH3
H
HO
H
O
H H
Pregnenolone
O
H
O
H H
Isoprogesterone
O
H
O
H H
Progesterone
O
H
O
H H
H
5¦Â-pregnan-3,20-dione
HO
H
O
H H
H
3¦Â-hydroxy-5¦Â-pregnan-20-one
( pregnanolone )
HO
H
O
H OH
H
3¦Â,14¦Â-dihydroxy-5¦Â-pregnan-20-one
HO
H
H OH
H
O O
Digitoxigenin
HO
H
H OH
H
O O
OH
Gitoxigenin
HO
H
H OH
H
O O
HO
Digoxigenin
O
H
H OH
H
O O
Dig-Dig-Dig-
O
H
H OH
H
O O
Glc-Dig-Dig-Dig-
Digitoxin
O
H
H OH
H
O O
Dig-Dig-Dig-
HO
O
H
H OH
H
O O
Glc-Dig-Dig-Dig-
OH
Digoxin
Desacetyl-lanatoside C
O
H
H OH
H
O O
Dig-Dig-Dig-
OH
O
H
H OH
H
O O
Glc-Dig-Dig-Dig-
OH
GitoxinPurpureaglycoside B
Farnesyl diphosphate
O PP
Campesterol
HO
24-Ethylidene lophenol
HO
CH3
HC
Isofucosterol
HO
HC
DXS DXR MCT CMK MDS HDS
HDR
HMGR MK PMK MPD
FDPS1
SQS1
SQS1
SMO
CAS1
SMT1 CPI1 CYP51G1 FK
HYD1
SMT2STE1DWF5
DWF1
CYP710A
STE1
DWF5DWF1
SCCE SCCE
3¦Â-HSD
3¦Â-HSD 5¦Â-POR 3¦Â-HS-5¦Â-OR mono-oxygenases?
Dp21MaT
Purpureaglycoside A
3-KSI
CGH I GT/UGT
GT/UGT
GT/UGT
O PP
Dimethylallyl diphosphate
Geranyl diphosphate
O PP
GPPS
IDI
Acetyl-CoA
S-CoA
O
S-CoA
O O
Acetoacetyl-CoA
HMGS
AACT
HO
H
HOH2C O
H OH
H
HO
H
O
H OH
H
O
OH
O
O
pregnane hydroxylases ?
3¦Â,14¦Â, 21-trihydroxy-5¦Â -pregnan-20-one21-O-malonyl 5¦Â-pregnan-3¦Â,14¦Â-dihydroxy-20-one
D12H
D12H
D12H
CGH I
Terpenoid backbone
Steroids
Cardenolides
MEP
Pathway
MVA
Pathway
Additional file 8. The putative biosynthetic pathway of cardiac glycosides in
Digitalis Purpurea.
The pathway roughly comprises terpenoid backbone biosynthesis, steroid biosynthesis and cardenolide
biosynthesis three stages. The enzymes with corresponding unigenes are framed. Abbreviation:
DXS,1-deoxy-D-xylulose-5-phosphate synthase; DXR,1-deoxy-D-xylulose-5-phosphate reductoisomerase; MCT,
2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK, 4-diphosphocytidyl-2-C-methyl-D-erythritol
kinase; MDS,2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS,4-hydroxy-3-methylbut-2-en-1-yl
diphosphate synthase; HDR,4-hydroxy-3-methylbut-2-enyl diphosphate reductase; AACT,acetyl-CoA
C-acetyltransferase; HMGS,hydroxymethylglutaryl-CoA synthase; HMGR,hydroxymethylglutaryl-CoA reductase;
MK, mevalonate kinase; PMK, phosphomevalonate kinase; MPD, diphosphomevalonate decarboxylase;

2. IDI,isopentenyl-diphosphate delta-isomerase; GGPPS1,geranylgeranyl pyrophosphate synthase 1; FDPS1,farnesyl
diphosphate synthase 1; SQS1,squalene synthase 1; SMO,squalene monooxygenase; CAS1,cycloartenol synthase 1;
SMT1,sterol 24-C-methyltransferase; CPI1,cyclopropyl isomerase; CYP51G1,cytochrome P450, family 51,
subfamily A (sterol 14-demethylase); FK, delta14-sterol reductase; HYD1， cholestenol delta-isomerase; SMT2，
sterol methyltransferase 2; STE1， C-5 sterol desaturase; DWF5， sterol delta7 reductase ; DWF1，delta24-sterol
reductase; SCCE ， cholesterol monooxygenase (side-chain-cleaving); 3βHSD ， 3-beta-hydroxysteroid
dehydrogenase; 3-KSI， 5△ -3-ketosteroid isomerase; 5βPOR，progesterone 5β-reductase; 3βHS- P5βOR，
3β-hydroxysteroid 5β-oxidoreductase; Dp21MaT，21-hydroxypregnane 21-O-malonyltransferase; D12H，digitoxin
12β-hydroxylase; CGHⅠ，cardenolide 16-O-glucohydrolase; GT，glycosyltransferase/ glucuronosyltransferase;
UGT，UDP- glycosyltransferase/ glucuronosyltransferase.
AMIYA KUMAR GHOSH
DEPARTMENT OF PHARMACEUTICAL CHEMISTRY
EMAIL:amiyaghosh94@gmail.com
contact: 8910679352