Expression of human steroid hydroxylases in fission yeast [Elektronische Ressource] / C̆ălin-Aurel Drăgan
114 Pages
English
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Expression of human steroid hydroxylases in fission yeast [Elektronische Ressource] / C̆ălin-Aurel Drăgan

Downloading requires you to have access to the YouScribe library
Learn all about the services we offer
114 Pages
English

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Expression of human steroid hydroxylases infission yeastDissertationzur Erlangung des Gradesdes Doktors der Naturwissenschaftender Naturwissenschaftlich-Technischen Fakulta¨t IIIChemie, Pharmazie, Bio- und Werkstoffwissenschaftender Universita¨t des SaarlandesvonC˘alin-Aurel Dra˘ganSaarbru¨cken05.08.201009.12.2010Tag des Kolloquiums:Prof. Dr.-Ing. Stefan DiebelsDekan:PD Dr. Matthias BureikBerichterstatter:Prof. Dr. Elmar HeinzleProf. Dr. Volkhard HelmsVorsitz:Dr. Britta DieselAkad. Mitarbeiter:ContentsList of Figures viList of Tables viiAbbreviations viiiSymbols and variables ixNotes on nomenclature and style xAbstract xiZusammenfassung xiiScientific contributions xiii1 Introduction 11.1 Steroids as chemical entities . . . . . . . . . . . . 11.2 Adrenal steroids . . . . . . . . . . . . . . . . . . . 31.3 Clinical aspects of steroid biosynthesis . . . . . . 61.4 Steroid synthesis . . . . . . . . . . . . . . . . . . 91.4.1 Biocatalysis by P450s . . . . . . . . . . . . 91.4.2 Chemical synthesis . . . . . . . . . . . . . 181.5 The rationale for this work . . . . . . . . . . . . . 201.5.1 Focus on recombinant whole-cell biotrans-formation . . . . . . . . . . . . . . . . . . 20iii1.5.2 Use of human P450s and fission yeast . . . 221.6 Aims of this work . . . . . . . . . . . . . . . . . . 272 Discussion 282.1 Functional expression of human CYP11B1 in fis-sion yeast . . . . . . . . . . . . . . . . . . . . . . 282.1.

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Published 01 January 2010
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Expression of human steroid hydroxylases in
fission yeast
Dissertation
zur Erlangung des Grades
des Doktors der Naturwissenschaften
der Naturwissenschaftlich-Technischen Fakulta¨t III
Chemie, Pharmazie, Bio- und Werkstoffwissenschaften
der Universita¨t des Saarlandes
von
C˘alin-Aurel Dra˘gan
Saarbru¨cken
05.08.201009.12.2010Tag des Kolloquiums:
Prof. Dr.-Ing. Stefan DiebelsDekan:
PD Dr. Matthias BureikBerichterstatter:
Prof. Dr. Elmar Heinzle
Prof. Dr. Volkhard HelmsVorsitz:
Dr. Britta DieselAkad. Mitarbeiter:Contents
List of Figures vi
List of Tables vii
Abbreviations viii
Symbols and variables ix
Notes on nomenclature and style x
Abstract xi
Zusammenfassung xii
Scientific contributions xiii
1 Introduction 1
1.1 Steroids as chemical entities . . . . . . . . . . . . 1
1.2 Adrenal steroids . . . . . . . . . . . . . . . . . . . 3
1.3 Clinical aspects of steroid biosynthesis . . . . . . 6
1.4 Steroid synthesis . . . . . . . . . . . . . . . . . . 9
1.4.1 Biocatalysis by P450s . . . . . . . . . . . . 9
1.4.2 Chemical synthesis . . . . . . . . . . . . . 18
1.5 The rationale for this work . . . . . . . . . . . . . 20
1.5.1 Focus on recombinant whole-cell biotrans-
formation . . . . . . . . . . . . . . . . . . 20
iii1.5.2 Use of human P450s and fission yeast . . . 22
1.6 Aims of this work . . . . . . . . . . . . . . . . . . 27
2 Discussion 28
2.1 Functional expression of human CYP11B1 in fis-
sion yeast . . . . . . . . . . . . . . . . . . . . . . 28
2.1.1 ThehumanCYP11B1isexpressedandcor-
rectly localized in fission yeast cells . . . . 29
2.1.2 Fission yeast strains expressing the human
enzyme CYP11B1 convert11-deoxycortisol
to cortisol in vivo . . . . . . . . . . . . . . 30
2.1.3 Space-time yield on cortisol . . . . . . . . 32
2.1.4 Fission yeast electronically sustains mito-
chondrial P450 reactions . . . . . . . . . . 34
2.1.5 The human CYP11B1 and CYP11B2 show
different kinetic properties when expressed
in fission yeast . . . . . . . . . . . . . . . 36
2.1.6 Application of CYP11B1 expressing fission
yeast strains for inhibition studies . . . . . 40
2.2 Functional expression of the microsomal human
P450s CYP17A1 and CYP21A1 in fission yeast . 41
2.2.1 Expression of microsomal P450s . . . . . . 41
2.2.2 Human CYP17A1 and CYP21A1 are func-
tionally expressed in fission yeast . . . . . 42
2.2.3 ApplicationofCYP17A1andCYP21A1ex-
pressing fission yeast strains for inhibition
studies . . . . . . . . . . . . . . . . . . . . 45
2.2.4 ElectroniccouplingofmicrosomalP450sto
host systems and its implications . . . . . 47
Conclusions 51
List of research papers in chronological order 52
ivAcknowledgments 54
A Proteins, peptides, genes, and DNA sequences 55
B Dragan et al. (2005) 56
C Dragan et al. (2006a) 62
D Dragan et al. (2006b) 79
Bibliography 90
vList of Figures
1.1 Chemical structure of lanosterol.. . . . . . . . . . 2
1.2 Ring designations and atom numbering in steroids 2
1.3 An exemplary conformer of cholesterol . . . . . . 3
1.4 Histology of the human adrenal . . . . . . . . . . 4
1.5 Steroid biosynthesis pathways in the human adrenal 5
1.6 Topology and structure of P450s. . . . . . . . . . 11
1.7 Hypothetical P450 reaction cycle . . . . . . . . . 13
1.8 Starting structures for partial steroid syntheses . 19
2.1 Modification of a 1.5 mL tube to yield a tip-tube 41
viList of Tables
1.1 Taxonomy of fission yeast . . . . . . . . . . . . . 23
2.1 Fission yeast strains used in this work. . . . . . . 29
A.1 Proteins,peptides,genes,andDNAsequencesused
in this work . . . . . . . . . . . . . . . . . . . . . 55
viiAbbreviations
Abbreviation Representation
16Prog 16 -hydroxyprogesterone
17Prog 17 -hydroxyprogesterone
18B 18 -hydroxycorticosterone
aa amino acid(s)
Aldo aldosterone
B corticosterone
bp base pair
Bp byproduct
CAH congenital adrenal hyperplasia
cDNA complementary DNA
CHO Chinese hamster ovary, cell line
CMV cytomegalovirus
COS African green monkey CV-1 cell line carrying SV-40
CBR cytochrome b reductase5
CPR cytochrome P450 reductase
CYP cytochrome P450
cyt b cytochrome b5 5
DAD diode array detector
DHEA dehydroepiandrosterone
DISC discontinuous
DNA desoxyribonucleic acid
DOC 11-deoxycorticosterone
EMM Edinburgh Minimal Medium
F cortisol
Fdx ferredoxin
GRAS generally regarded as safe
HPLC high pressure liquid chromatography
HSD hydroxysteroid dehydrogenase
IUB International Union of Biochemistry
IUPAC International Union of Pure and Applied Chemistry
LLE liquid-liquid extraction
NAD nicotinamid adenine dinucleotide
NADP nicotinamid adenine dinucleotide phosphate
NCYC National Collection of Yeast Cultures
nt nucleotide
P product
PAGE polyacrylamide gel electrophoresis
PPP pentose phosphate pathway
Prog progesterone
ROS reactive oxygen species
S substrate
SDS sodium dodecyl sulfate
SREBP sterol regulatory element binding protein
TLC thin layer chromatography
V79 Chinese hamster lung fibroblast cell line
viii
abaSymbols and variables
Abbreviation Representation Unit
c concentration M
c(t) concentration at time t M
−1hCRi average space-time yield M d
-2g earth’s centripetal acceleration m s
IC inhibitor concentration at which 50 % activity M50
K pseudo-Michaelis-Menten constant M1/2
λ wavelength nm
t time d
−1v maximum conversion velocity M smax
ix
?
?
?Notes on nomenclature and
style
All nomenclature uses accepted standard symbols as far as pos-
sible. Units are typeset according to SI. Element nomenclature
follows IUPAC rules. Biochemical nomenclature follows IUB rec-
ommendations.
Carbon atoms belonging to a molecule are addressed as ’C-
n’ where ’C’ stands for carbon and n denotes the atom number
according to IUPAC rules. The total number of carbon atoms
of a molecule is abbreviated as ’C ’ with ’C’ again standing forn
carbon and n being the carbon atom count.
A redox reaction of the form X +Y −→ X +Y is oftenox oxred red
additionally given in a simplified form as X−→ Y to emphasize
the vectorial flow of electrons.
Enzymenamesareoftentypesetas’ Protein’with’s’denotings
the source species where ’s’ can be substituted by either ’b’ for
bovine,’h’forhuman,or’r’forratproteins. Fissionyeastproteins
are not labeled by a specific prefix subscript. Species names as
wellasgenenamesaretypesetinitalicsaccordingtogeneralrules.
x