Академический Документы
Профессиональный Документы
Культура Документы
Lloyd, 1910
Imbert, 1998
Aryltetralin lignan derivatives and
abbreviations
b-Peltatin H OH OCH3
(1) (2)
pET/15b pYes2/NT C
Vector Vector
BL21(DE3)pLysS INVSc1
Heterologous Heterologous
protein purification protein purification
(-)
(+)
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
Km NADPH: 43.7 µM
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
• 10 µl KCN 0.8
• 5 µl microsomes 0.7
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
• t: time in minutes 0 2 4 6 8
TIME (MIN)
10 12 14
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)
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)
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!