Identification of genes involved in the

biosynthesis of lignans in Linum flavum

Supervisor: Prof. Dr. Maike Petersen

Introduction
• Linum flavum (golden flax,
yellow flax)
• Family Linaceae, native to
central and southern Europe
• Erect, woody perennial, growing
to 30 cm tall
• Bright yellow, five-petalled
flowers
• Accumulation of lignans
Lignans - Biological activity
• Defense against pathogens and infections
• Antimycotic, antiviral and antibacterial
• Laxative, cathartic, anthelmintic, antidote for snakebites
• Emetic and cholagogue

Lloyd, 1910
Imbert, 1998
Aryltetralin lignan derivatives and
abbreviations

• Podophyllotoxin (PTOX) strongly cytotoxic

• PTOX first isolated from roots and rhizomes
of plants of the genus Podophyllum
• PTOX binds to the α/β tubulin dimer and
inhibits the construction of microtubules R1 R2 R3
Podophyllotoxin OH H OCH3
6-Methoxy podophyllotoxin OH OCH3 OCH3
Deoxypodophyllotoxin H H OCH3
a-Peltatin H OH OH

b-Peltatin H OH OCH3

ß-Peltatin A methyl ether H OCH3 OCH3

Biosynthesis of lignans - Early steps

(1) (2)

From coniferyl alcohol to matairesinol

(1) pinoresinol synthase
(2)
(2) pinoresinol/lariciresinol reductase
(3) secoisolariciresinol dehydrogenase (3)
Biosynthesis of lignans
- Late steps

• Lignan biosynthetic pathways downstream

of matairesinol
• Different models and hypotheses

Marques et al., 2013

Biosynthesis of lignans - Late steps

Lau and Sattely, 2015

Workflow
gDNA Phusion-
PCR
PCR
pDrive Full-length
Vector sequence
RNA cDNA PCR

pET/15b pYes2/NT C
Vector Vector

BL21(DE3)pLysS INVSc1

Heterologous Heterologous
protein purification protein purification

Enzyme assay Enzyme assay

1. Project: Identification and characterization
of pinoresinol-lariciresinol reductase (PLR)
Pinoresinol-lariciresinol reductase (PLR)
• EC 1.23.1.1
• Bifunctional
• different stereospecificity

Heimendahl et al., 2005

PLR - Candidat

Blastp with PLR from Linum usitatissimum

Contig Score Query E value Identity

cover

10318 544 93% 0.0 83%

Sequence of candidate was obtained from https://db.cngb.org/onekp/

Ligation into pET-15b
Western-blot

MW of candidate appr. 38 kDa

(including His-tag)

M: marker; D: flow through fraction; W1: wash

fraction 1; W2: wash fraction 2; W6: wash fraction
6; E1: elution fraction 1; E2: elution fraction 2
PLR enzyme assay
• 6 µl protein preparation (0.35 µg/ml)
• 6 µl 5 mM racemic pinoresinol
• 0.1-100 µl 2.5 mM NADPH
• KPi-buffer (5.5-9) ad 250 µl

• incubated at different temperatures

• Extract with 3X 500 µl ethyl acetate
• Evaporate in vacuum centrifuge
• Redissolving: 100 µl methanol
 HPLC
HPLC conditions: column Hypersil HyPurityTM Elite C18 4.6 mm; water:acetonitrile 75:25; flow rate:
1.5 ml/min; wavelength: 280 nm

Reference secoisolariciresinol Reference pinoresinol

HPLC
0’ 1’ 2’ 5’ 10’ 20’ 30’
 Chiral column HPLC
HPLC conditions: column Chiracel OD-H; ethanol:n-hexane 50:50;
flow rate: 0.6 ml/min; wavelength: 280 nm (-) (-)
(-)(+)

(-)
(+)

Reference racemic
Reference racemic pinoresinol 30’
secoisolariciresinol
 PLR – optimal conditions

pH Temperature
300 250
250 200

(nmol.min-1.mg-1)
(nmol.min-1.mg-1)

Specific Activity
Specific Activity

200
150
150
100
100
50 50
0 0
5.5 6 6.5 7 7.5 8 8.5 9 21 24 27 30 33 36 39 42
pH Temperature (°C)
 CPR – saturation kinetics
NADPH Hanes-Woolf plot – PLR - NADPH
350 3

S/v (µM.min.mg.nmol-1)
300 y = 0.0033x + 0.1444
(nmol.min-1.mg-1)

2.5
Specific Activity

250
2
200
1.5
150
100 1
50 0.5
0 0
0 200 400 600 800 0 200 400 600 800

Concentration NADPH (µM) Concentration NADPH (µM)

Km NADPH: 43.7 µM

Vmax: 303 nmol.min-1.mg-1

2. Project: Identification of genes encoding
Cytochrome P450 reductase
 Cytochrome P450 reductase (CPR)
• Cytochrome P450 reductase (EC 1.6.2.4; also referred as
NADPH:ferrihemoprotein oxidoreductase,
NADPH:hemoprotein oxidoreductase, NADPH:P450
oxidoreductase, P450 reductase, POR, CPR, CYPOR)
• localized in the endoplasmic reticulum
• In animals, only one CPR interacts with all cytochrome
P450s.
• In plants multiple CPR isoforms exist.

http://www.cchem.berkeley.edu/clarkgrp/GroupMembersPages/MattTraylor.html
 CPR-Candidates
• Partial sequence of NADPH:cytochrom P450-reductase (CPR) from Linum flavum (Kuhlmann,
Dissertation, Philipps-Universität Marburg, 2004)
• Transcriptome of Linum flavum: http://www.medplantrnaseq.org/
• Blastn: http://medplants.ncgr.org/quorum/jobs

Contig Score Query cover E value Identity

66401 610 100% 7e-141 98%

5729 587 100% 0.0 94%

4753 487 100% 0.0 70%

5254 401 100% 7e-140 70%

CPR – ligation into pYes2/NTC
CPR – Western Blot

MW of 4753 is circa 79 kDa.

Dyeing with Coomassie brilliant blue
MW of 66401 is circa 77 kDa.
 CPR – enzyme assay PHOTOMETRICAL ENZYME ASSAY

• 10 µl KCN 0.8

• 5-90 µl 2 mM cytochrome c 0.75

• 5 µl microsomes 0.7

• 765-964 µl 0.5 mM Tris/HCl pH 7.6

ABSORPTION AT 550 NM
0.65

• 1-200 µl 6 mM NADPH
0.6
Sample
final volume 1 ml Negative control
0.55
photometrically monitored at 550 nm
0.5

Specific activity = A550/t • ε550 • csample 0.45

Addition of NADPH
• A550: absorption difference
0.4

• t: time in minutes 0 2 4 6 8
TIME (MIN)
10 12 14

• ε550 : 21 mM-1cm-1 ; absorption coefficient of cytochrome

c at 550 nm
• csample: concentration of protein
 CPR – saturation kinetics
Substrate saturation curve cyt. c - Hanes Woolf Plot cyt. c - 66401
Specifice Activity (nmol.min-1.mg-1)

0.8
66401 0.7
y = 0.0034x + 0.0277
R² = 0.9912

S/v (µM.min.mg.nmol-1)
350.00
300.00
0.6 Km cyt.c: 8.15 µM
0.5
250.00
0.4
200.00
150.00 0.3
Vmax: 294 nmol.min-1.mg-1
100.00 0.2
50.00
0.1
0.00
0 50 100 150 200 0
-50 -0.1 0 50 100 150 200 250
Concentration Cytochrome c (µM)
Concentration Cytochrome c (µM)

Substrate saturation curve NADPH - Hanes Woolf Plot NADPH - 66401

66401
Specifice Activity (nmol.min-1.mg-1)

5
y = 0.0038x + 0.1126
Km NADPH: 29.6 µM
S/v (µM.min.mg.nmol-1)
300 4 R² = 0.9984
250
3
200
150
2 Vmax: 263 nmol.min-1.mg-1
100 1

50 0
0 -200 0 200 400 600 800 1000 1200 1400
-1
0 200 400 600 800 1000 1200 1400 Concentration NADPH (µM)
Concentration NADPH (µM)
 CPR – saturation kinetics
Substrate saturation curve cyt. c - 4753 Hanes Woolf Plot cyt.c - 4753
350.00 0.8
y = 0.0034x + 0.0529
Specifice Activity (nmol.min-1.mg-1)

S/v (µM.min.mg.nmol-1)
300.00 0.7 R² = 0.9838
0.6
250.00
200.00
0.5 Km cyt.c: 15.6 µM
0.4
150.00 0.3
100.00 0.2 Vmax: 294 nmol.min-1.mg-1
50.00 0.1
0
0.00
-50 0 50 100 150 200 250
0 50 100 150 200
Concentration Cytochrom c (µM)
Concentration Cytochrom c (µM)

Substrate saturation curve NADPH

Hanes Woolf Plot NADPH - 4753
- 4753
Specifice Activity (nmol.min-1.mg-1)

3.5
500

S/v (µM.min.mg.nmol-1)
450 3 y = 0.0026x + 0.1174
400
350 2.5
R² = 0.9962 Km NADPH: 45.15 µM
300 2
250
200 1.5
150
100 1
50 Vmax: 384 nmol.min-1.mg-1
0 0.5
0 200 400 600 800 1000 1200 1400 0
Concentration NADPH (µM) -200 0 200 400 600 800 1000 1200 1400
Concentration NADPH (µM)
Literature
• Benveniste et al., 1991. Biochem Biophys Res Commun 177: 105-112
• Bonina et al., 2005. Biochemistry 70: 357–365
• Chu et al., 1993. J. Biol. Chem. 268: 27026-27033
• Eberle et al., 2009. Plant Mol Biol 69: 239-253
• Federolf et al., 2007. Phytochemistry 68: 1397-1406 274, 618 -627
• Fujita et al.,1999. J. Biol. Chem.
• Heimendahl et al., 2005. Phytochemistry 66: 1254–1263
• Kleinig H. and Mayer U., 1999. Zellbiologie, G. Fischer Verlag
• Kranz, 2004. Dissertation, Philipps-Universität Marburg
• Kuhlmann, S., 2004. Dissertation, Philipps-Universität Marburg
• Lau, W., Sattely, E.S., 2015. Science 349: 1224-1228
Literature
• Marques JV et al., 2013. J Biol Chem 288(1): 466-79
• Lloyd, 1910. Bulletin of the Lloyd
• Imbert, 1998. Biochemie 80: 207-222
• Lu et al., 1969. The Journal of Biological Chemistry 244 (13): 3714–3721
• Molog et al., 2001. Planta 214: 288-294
• Ro DK et al., 2002. Plant Physiol 130: 1837-1851
• Omura T., Sato R., 1964. J. Biol. Chem. 239: 2370-2378
• Wang et al., 1997. Proc Natl Acad Sci USA 94: 8411–8416
• Werck-Reichart et al., 2002. The Arabidopsis Book
Thank you for your attention!