Malonylcoenzyme A:21-hydroxypregnane-21-O-malonyltransferases from Digitalis sp. [Elektronische Ressource] / vorgelegt von Serge Philibert Kuate

De
Malonylcoenzyme A: 21-hydroxypregnane-21-O-malonyltransferases from Digitalis sp. Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades vorgelegt von Serge Philibert Kuate aus Kamerun Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 17.09.2008 Vorsitzender der Promotionskommission: Prof. Dr. Eberhard Bänsch Erstberichterstatter: Prof. Dr. Wolfgang Kreis Zweitberichterstatter: Prof. Dr. Georg KreimerSP Kuate (2008): Doctoral Thesis „Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes“ SP Kuate (2008): Doctoral Thesis Dedication To my Dad, my Mum, my Brothers and Sisters, my whole Family, may this be an element of pride. This is a special reward for my Dear Spouse Bernice and our Daughter Carelle, for their support and for have tenderly enduring my regular and long absences. SP Kuate (2008): Doctoral Thesis Contents Contents Contents................................................................................................................................. i Acknowledgements........................................................................................................
Publié le : mardi 1 janvier 2008
Lecture(s) : 34
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Source : WWW.OPUS.UB.UNI-ERLANGEN.DE/OPUS/VOLLTEXTE/2008/1112/PDF/SERGEKUATEDISSERTATION.PDF.PDF
Nombre de pages : 182
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Malonylcoenzyme A:
21-hydroxypregnane-21-O-malonyltransferases from Digitalis sp.








Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades








vorgelegt von
Serge Philibert Kuate
aus Kamerun

















Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der
Universität Erlangen-Nürnberg





Tag der mündlichen Prüfung: 17.09.2008

Vorsitzender der
Promotionskommission: Prof. Dr. Eberhard Bänsch

Erstberichterstatter: Prof. Dr. Wolfgang Kreis

Zweitberichterstatter: Prof. Dr. Georg Kreimer
SP Kuate (2008): Doctoral Thesis




























„Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes“
SP Kuate (2008): Doctoral Thesis

Dedication





To

my Dad, my Mum,
my Brothers and Sisters,
my whole Family,


may this be an element of pride.





This is a special reward for
my Dear Spouse Bernice and our Daughter Carelle,
for their support and for have tenderly enduring
my regular and long absences.
SP Kuate (2008): Doctoral Thesis Contents

Contents

Contents................................................................................................................................. i
Acknowledgements............................................................................................................. vi
List of abbreviations..........................................................................................................vii
List of figures ...................................................................................................................... ix
List of tables xiv

Chapter I: Introduction ...................................................................................................... 1

I.1. Digitalis and Isoplexis sp. ........................................................................................... 1
I.1.1. Morphology and distribution................................................................................ 1
I.1.2. Systematic classification ...................................................................................... 2
I.1.3. Constituents and medicinal uses of Digitalis and Isoplexis sp. ........................... 3
I.1.4. Heart diseases....................................................................................................... 3

I.2. Cardiac glycosides 4
I.2.1. Structure ............................................................................................................... 4
I.2.2. Biochemical mechanism of action ....................................................................... 7
I.2.3. Biosynthesis of cardiac glycosides 8

I.3. Enzymes .................................................................................................................... 11
I.3. Transferases........................................................................................................... 11
I.3.1. Malonyltransferases ........................................................................................... 12
I.3.2. Malonyltransferases in flavonoids metabolism.............................................. 13
I.3.3. Malonyltransferases in fatty acids metabolism 14
I.3.4. Malonyltransferases in other metabolic pathways ......................................... 14

I.4. Problematic and objectives........................................................................................ 16

Chapter II: Materials and Methods................................................................................. 18

II.1. Development of a suitable method for the quantification of the malonylcoenzyme A:
21-hydroxypregnane 21-O-malonyltransferase reaction................................................. 18
II.1.1. Synthesis of a putative substrate for the 21MaT............................................... 18
II.1.1.1. Reagents and chemicals ............................................................................. 18
II.1.1.2. Synthesis of 3β-benzoyloxy-5β-pregnane-14β,21-dihydroxy-20-one (5) . 18
II.1.1.2.a. Synthesis of benzoyldigitoxigenin (4) ................................................ 18
II.1.1.2.b. Synthesis of 3β-benzoyloxy-5β-pregnan-14β, 21-dihydroxy-20-one
(5). ....................................................................................................................... 19
II.1.1.2.c. Synthesis of 3β-benzoyloxy-5β-pregnan-14β-hydroxy-20-on-21-
malonylhemiester (6)........................................................................................... 20
II.1.1.3. Synthesis of 3β-acetoxy-5β-pregnan-14β, 21-dihydroxy-20-one (1) ........ 21
II.1.2. Detection of the synthesized compounds.......................................................... 21
II.1.2.1. Anisaldehyde reagent................................................................................. 21
II.1.2.2. Tetrazolium blue reagent ........................................................................... 22
II.1.2.3. Kedde reagent ............................................................................................ 22
II.1.3. Characterization of the synthesized products ................................................... 22
SP Kuate (2008): Doctoral Thesis i Contents
II.1.3.1. Enzymatic and chromatographic characterizations ................................... 22
II.1.3.2. Spectral characterization............................................................................ 23
II.1.4. Establishment of the HPLC method ................................................................. 23
II.1.2.1. Instrumentation and operating conditions.................................................. 23
II.1.2.2. UV spectra of the synthesized malonylated products................................ 24
II.1.2.3. Chromatographic separation and linearity................................................. 24
II.1.2.4. Sensibility and variability of the method................................................... 24
II.1.2.5. Application................................................................................................. 25

II.2. Purification of malonylcoenzyme A: 21-hydroxypregnane 21-O-malonyltransferase
from D. purpurea and D. lanata...................................................................................... 25
II.2.1. Chemicals.......................................................................................................... 25
II.2.2. Plant material .................................................................................................... 26
II.2.3. Protein quantification........................................................................................ 26
II.2.3.1. Bradford Assay .......................................................................................... 26
II.2.3.2. Preparation of the reagent .......................................................................... 26
II.2.4. Standard enzyme assay for the 21MaT............................................................. 27
II.2.5. Chitin azure chitinase assay.............................................................................. 28
II.2.6. Flavonoid assay................................................................................................. 28
II.2.7. Enzyme purification from D. purpurea (Dp21MaT)........................................ 29
II.2.7.1. Preparation of the crude extract ................................................................. 29
II.2.7.2. Ammonium sulphate precipitation 30
II.2.7.2.a. Principle .............................................................................................. 30
II.2.7.2.b. Experiment 31
II.2.7.3. Hydrophobic interaction chromatography on Phenylsepharose 6 FF........ 31
II.2.7.3.a. Principle 31
II.2.7.3.b. Experiment.......................................................................................... 32
II.2.7.4. Anion exchange chromatography on Source 30 Q .................................... 32
II.2.7.4.a. Principle 32
II.2.7.4.b. Experiment 32
II.2.7.5. Affinity chromatography on Cibacron Blue F3G-A.................................. 33
II.2.7.5.a. Principle .............................................................................................. 33
II.2.7.5.b. Experiment 33
II.2.7.6. Gel filtration chromatography on Superdex 75 10/300 GL....................... 34
II.2.7.6.a. Principle 34
II.2.7.6.b. Column calibration ............................................................................. 34
II.2.7.6.c. Gel filtration as purification step ........................................................ 35
II.2.7.7. Others chromatographic columns .............................................................. 35
II.2.7.7.a. Octylsepharose FF column.................................................................. 35
II.2.7.7.b. Butylsepharose FF column ................................................................. 35
II.2.7.7.c. HiTrap DEAE FF column ................................................................... 36
II.2.7.7.d. SP Sepharose FF column .................................................................... 36
II.2.7.7.e. Superose 12 ......................................................................................... 36
II.2.8. Enzyme purification from D. lanata (Dl21MaT). ............................................ 36

II.3. Characterization of malonylcoenzyme A: 21-hydroxypregnane 21-O-
malonyltransferase........................................................................................................... 37
II.3.1. Temperature optimum....................................................................................... 37
II.3.2. pH optimum ...................................................................................................... 37
II.3.3. Enzyme stability ............................................................................................... 38
SP Kuate (2008): Doctoral Thesis ii Contents
II.3.4. Effect of divalent cations .................................................................................. 38
II.3.5. Acyl-donor specificity ...................................................................................... 39
II.3.6. Acyl-acceptor specificity 39
II.3.6.1. Steroid substrates with 21-OH................................................................... 39
II.3.6.2. Steroid substrates with OH groups positioned at different positions......... 39
II.3.6.3. Flavonoid substrates................................................................................... 40
II.3.7. Polyacrylamide gel electrophoresis (PAGE) .................................................... 41
II.3.7.1. Principle ..................................................................................................... 41
II.3.7.2. Experiment................................................................................................. 42
II.3.8. Preparative PAGE and electroelution ............................................................... 44
II.3.8.1. Principle 44
II.3.8.2. Experiment 44
II.3.9. Isoelectric focusing ........................................................................................... 46
II.3.9.1. Principle 46
II.3.9.2. Experiment 47
II.3.10. Two-dimensional electrophoresis (2-D PAGE).............................................. 48
II.3.10.1. Isoelectric focusing .................................................................................. 48
II.3.10.1.a. Sample preparation............................................................................ 48
II. 3.10.1.b. Experiment....................................................................................... 49
II.3.10.2. SDS-PAGE .............................................................................................. 49
II.3.11. Staining methods............................................................................................. 50
II.3.11.1. Silver staining .......................................................................................... 50
II.3.11.1. Coomassie blue staining .......................................................................... 51
II.3.12. Protein sequencing and identification by mass spectrometry......................... 52
II.3.12.1. In gel digestion......................................................................................... 52
II.3.12.2. NanoLC-MS/MS and De Novo sequencing ........................................... 53

II.4. Immunological aspects 53
II.4.1. Chemicals.......................................................................................................... 53
II.4.2. Production of polyclonal antibodies against the purified enzyme.................... 54
II.4.3. Antiserum titer determination by ELISA.......................................................... 54
II.4.4. Western blot...................................................................................................... 55
II.4.4.1. Principle ..................................................................................................... 55
II.4.4.2. Experiment................................................................................................. 56
II.4.5. Antibody inhibition test .................................................................................... 57

Chapter III: Results 58

III.1. Synthesis of 3β-benzoyloxy-5β-pregnane-14β,21-dihydroxy-20-one (5).............. 58
III.1.1. Synthesis of benzoyldigitoxigenin (4)............................................................. 58
III.1.2. Synthesis of 3β-benzoyloxy-5β-pregnane-14β, 21-dihydroxy-20-one (5)...... 59
III.1.3. Synthesis of 3β-benzoyloxy-5β-pregnane-14β-hydroxy-20-on-21-malonyl-
hemiester (6)................................................................................................................ 59
III.1.4. Characterization of synthesized products........................................................ 60

III.2. Establishment of the quantification method ........................................................... 65
III.2.1. UV spectra of 2 and 6..................................................................................... 65
II.2.2. Chromatographic separation and linearity 67
III.2.3. Precision and sensibility.................................................................................. 67

SP Kuate (2008): Doctoral Thesis iii Contents
III.3. Purification and characterization of the 21MaT..................................................... 69
III.3.1. Evaluation of potential starting materials........................................................ 69
III.3.1. Purification of the 21-hydroxypregnane-21-O-malonytransferase from D.
purpurea L. (Dp21MaT) ............................................................................................. 71
III.3.1.1. Ammonium sulphate precipitation ........................................................... 71
III.3.1.1. Hydrophobic interaction chromatography (HIC) on Phenylsepharose 6 FF
................................................................................................................................. 72
III.3.1.2. Anion exchange chromatography on Source 30 Q................................... 73
III.3.1.3. Affinity chromatography on Cibacron Blue F3G-A FF ........................... 74
III.3.1.3. Gel filtration on Superdex 75 ................................................................... 75
III.3.2. Purification of the 21-hydroxypregnane-21-O-malonytransferase from D.
lanata Ehrh. (Dl21MaT).............................................................................................. 76
III.3.2.1. Ammonium sulphate precipitation ........................................................... 76
III.3.2.2. Hydrophobic interaction chromatography on Phenylsepharose............... 77
III.3.2.3. Anion exchange chromatography on Source 30 Q................................... 78
III.3.2.4. Size exclusion chromatography on Superdex 75 10/300 GL ................... 79

III.4. 21-hydroxypregnane-21-O-malonytransferase: characterization....................... 80
III.4.1. Enzyme identification.................................................................................. 80
III.4.1.1. Identification of the 21-hydroxypregnane-21-O-malonytransferase from
D. purpurea L. ..................................................................................................... 81
III.4.1.2. Identification of the 21-hydroxypregnane-21-O-malonyltransfe-rase
from D. lanata Ehrh. ........................................................................................... 83
III.4.2. Determination of the molecular mass of the 21MaT................................... 85
III.4.2.1. Molecular mass determination using gel filtration 75 10/300 GL........ 85
III.4.2.2. Molecular mass determination using SDS-PAGE................................ 86
III.4.3. Time dependence......................................................................................... 88
III.4.4. Temperature and pH optima........................................................................ 88
III.4.4.1. Temperature optimum .......................................................................... 88
III.4.4.2. pH optimum.......................................................................................... 90
III.4.5. Enzyme stability 90
III.4.5.1. Protein concentration and enzyme stability at 4°C............................... 91
III.4.5.2. Enzyme stability at different temperatures........................................... 93
III.4.6. Effect of divalent cations............................................................................. 94
III.4.7. Acyl-donor specificity ................................................................................. 95
III.4.8. Acyl-acceptor specificity 97
III.4.8.1. Steroid substrates with 21-OH.................................................................. 97
III.4.8.2. Steroid substrates with OH groups at different positions......................... 99
III.4.8.3. Flavonoid substrates ............................................................................... 100
III.4.9. Isoelectric point ............................................................................................. 102
III.4.10. Partial peptide sequencing of the 21MaT................................................ 103
III.4.11. Chitinase assay ........................................................................................ 106

III.5. Production of polyclonal antibodies..................................................................... 107
III.5.1. Isolation of the Dl21MaT .............................................................................. 107
III.5.2. Effectiveness of the immunization ................................................................ 108

Chapter IV: Discussion ................................................................................................... 110

IV.1. Selection of the starting materials for the purification of the 21MaT .............. 110
SP Kuate (2008): Doctoral Thesis iv Contents
IV.2. Purification of malonylcoenzyme A: 21-hydroxypregane 21-O-
malonyltransferase from D. purpurea L. (Dp21MaT). ............................................. 112

IV.3. Purification of malonylcoenzyme A: 21-hydroxypregane 21-O-
malonyltransferase from D. lanata Ehrh. (Dl21MaT)............................................... 115

IV.4. Characterization of the malonylcoenzyme A: 21-hydroxypregane 21-O-
malonyltransferase..................................................................................................... 119

IV.5. Antibody production ........................................................................................ 125

IV.6. Partial peptide sequencing................................................................................ 126

Summary .......................................................................................................................... 130

Zusammenfassung ........................................................................................................... 132

V. References.................................................................................................................... 134

VI. Appendix 146
























SP Kuate (2008): Doctoral Thesis v Acknowledgements
Acknowledgements

I would like to thank my PhD supervisor Prof. Dr. Wolfgang Kreis for giving me
the opportunity to join his research group and for proposing me this work, dealing
with a central question in the cardiac glycosides biosynthesis. I am grateful for its
support and intellectual stimulation.
I owe a big thank to Prof. Dr. Georg Kreimer from the chair of Plant Ecophysiology
who thanks his several reports and recommendations always readily sustained my
application for the fellowship extention.
I wish to thank sincerely my Dear Willi, Dr WF Eisenbeiß for have being a
wunderful mentor, for constant fruitful discussions and for the great time spent
togheter. I especially thank Dr F. Müller-Uri for its high cooperativity and its
perticular assistance during the search of a postdoctoral position.
I also want to thank all my lab mates: Rodrigo, Nadine, Pia, Steffi, Andreas, Juliane
for their constant support, their friendship and because my time in the lab would
have been less enjoyable without them. I thank my elder Vanessa and Jördis for
help readiness and nice discussions.
Many thanks to the technical assistance staff: Grudrun, Renate and Gabi. I would
definitely never forget regular and enjoyables football discussions and jokes with
the former. The assistance of Renate during immunogical assays is specially
acknowledged. Special thanks to my several undergraduates and graduates students.
I owe my parents Mr and Ms Taghuo a big thank for their love, their sacrifices
throughout my education since thirthy years; my step-mother for her kind
encouragements and love. I am grateful to my friends Jöel and Clarisse (Freiburg),
friends and DAAD collegues Poumale and Marcus (presently in Yaoundé), Sao
(Kassel), my several friends in Erlangen (Marcel, Emma and Williams, Vitalis,
Florence, etc.), Rouen Verron (Edea), Fernand (Dschang), etc. for their constant
friendship and kind support. I also owe thanks to my all my teachers, from the
primary school to the University (Dschang and Yaounde I). This is a modest fruit of
your efforts. Finally, I am especially grateful to the German Academic Exchange
Service (DAAD) for granting me a fellowship to carry out this study.
SP Kuate (2008): Doctoral Thesis vi

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