Characterization of three 2-hydroxy-acid dehydrogenases in the context of a biotechnological approach to short-circuit photorespiration [Elektronische Ressource] / vorgelegt von Martin Engqvist

Characterization of three 2-hydroxy-acid
dehydrogenases in the context of a
biotechnological approach to short-circuit
photorespiration




Inaugural-Dissertation
zur
Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Universität zu Köln

vorgelegt von

Martin Engqvist
Aus Dänningelanda, Schweden


Köln, 2010




Die dieser Dissertation zugrunde liegenden experinmtellen Arbeiten wurden
in der Zeit von September 2007 bis Märtz 2010 amta niBsochen Institut der
Universität zu Köln angefertigt und in Teilen lim 2 0J09 upublizier t.



Engqvist, M., Drincovich, MF., Flügge, UI. andin oM,a urVG.
Two D-2-hydroxyacid dehydrogenases iAnr abidopsis thaliana with catalytic capacities to
participate in the last reactions of the methxyallgl yaond β-oxidation pathways
Journal of Biological Chemistry (20809)4: 25026-25037.












Prüfungsvorsitzender: Prof. Dr. Reinhard Krämer

Berichterstatter: Prof. Dr. Ulf-Ingo Flügge
Prof. Dr. Sabine Waffenschmidt
Prof. Dr. Hermann Bauwe


Tag der mündlichen Prüfung: 02. Juni 2010
II






















“Why spend a day in the library when you canth el esaarmn e thing
by working in the laboratory for a mo nth?”

Frank H. Westheimer (1912–2007)
III Table of contents
Table of contents
1. Introduction ........................................................................................ 8
1.1 Photosynthesis .......................................................................................... .... 8
1.1.1 Light reactions ..................................................................................................... 8
1.1.2 The dark reactions .................................................................................................................. 8
1.2 Photorespiration ....................................................................................... .... 9
1.2.1 The photorespiratory pathway.............................................................................. 9
1.2.2 Physiological importance of photorespira..t.io..n.. ................................................... 12
1.2.3 Reasons for decreasing photorespiration ............................................................................ 12
1.2.4 C4 photosynthesis ............................................................................................ 13
1.3 Biotechnological approaches to reducing photsoprieration ................................. .1..3 .....
1.3.1 Improving RuBisCO ............................................................................................ 13
1.3.1.1 Heterologous expression of natural RuBi s.C..Os......................................................................... . 14
1.3.1.2 Rational design of RuBisCO ................................................................................... .... 15
1.3.1.3 Random mutagenesis of RuBisCO ............................................................................. .. 15
1.3.2 Artificial C4-photosynthesis .............................................................................. 15
1.3.3 Short-circuiting photorespiration ........................................................................ 16
1.3.3.1 Introducing the bacterial glycerate pa thinwtaoy chloroplasts ................................................... 16
1.3.3.2 Introducing a complete glycolate captabthowlaicy into chloroplasts............................... 17
1.3.3.3 Improving the GMK pathway ................................................................................. ... 18
1.3.3.4 Other 2-hydroxy-acid dehydrogenases ..................................................................... .. 19
1.4 Goals of the present study ............................................................................................. 21
2. Materials and methods ............................................................... ..2. 2....
2.1 Plant growth conditions ........................................................................... . 22
2.1.1 Soil composition and stratification ................................................................... 22
2.1.2 Greenhouse growth conditions ............................................................................................ 22
2.1.3 Growth chamber conditions .............................................................................. 22
2.1.4 BASTA selection on soil ................................................................................ 22
2.1.5 A. thaliana seed sterilization .......................................................................... 22
2.1.6 A. thaliana growth and selection on sterile plates..................................................... ..2.3.. .....
2.1.7 Growth of root cultures .................................................................................. 23
2.2 Escherichia coli strains and growth conditions.................................................. ..... 24
2.3 Molecular biology methods ....................................................................... 24
2.3.1 PCR ........................................................................................................................................ 24
IV Table of contents
2.3.2 Extraction oA.f thaliana genomic DNA ....................................................................... 24
2.3.3 Extraction oA.f thaliana RNA .......................................................................................5.. ...... 2
2.3.4 DNAse treatment of RNA and first-strand scyDnNtAh esis ......................................... 25
2.3.5 Isolation of homozygAo.u st haliana T-DNA insertion mutants .................................. 25
2.3.6 Confirmation of knock-out plants by RT..-. ..P..C..R. ................................................ 26
2.3.7 Producing TSS-competeEn.t coli cells .................................................................................. 27
2.3.8 Heat-shock transformation Eo.f coli cells ............................................................... 27
2.3.9 Isolation Eo. fc oli plasmid DNA ....................................................................... ..2.7.. ..
2.3.10 Separation of DNA by agarose gel electreospihso .r................................................ 27
2.3.11 DNA elution from agarose gels ........................................................................................... 28
2.3.12 DNA sequencing .............................................................................................. 28
2.3.13 Cloning of the three candidate enzym.e..s. ........................................................ 29
2.4 Biochemical methods ................................................................................ . 29
2.4.1 Expression and purification of recombinoatneti nps r.......................................................... 29
2.4.2 SDS-polyacrylamide gel electrophoresis A(GSED)S -.P............................................... 31
2.4.3 Protein determination and Coomassie sta.i.n..in..g. ................................................. 31
2.4.4 Western-Blot .................................................................................................. 32
2.4.5 Native-PAGE ......................................................................................................................... 32
2.4.6 Mitochondrial isolation .................................................................................... 33
2.4.6 Size exclusion chromatography ......................................................................... 33
2.4.7 Analysis of prosthetic groups .......................................................................... 33
2.5 Enzyme assays .............................................................................................................. .. 34
2.5.1 Co-factor analysis ........................................................................................... 34
2.5.2 pH optimum and substrate screen .................................................................. 34
2.5.3 Catalytic constants .......................................................................................... 35
2.6 In silico protein analysis .................................................................................................. 35
2.6.1 Phylogenetic trees ........................................................................................... 35
2.6.2 Co-expression analysis...................................................................................... 35
2.6.3 Catalytic site analysis ...................................................................................... 35
2.7 Metabolite and fluorescence measurement.s. ........................................................... .3. .6
2.7.1 Metabolite analysis by GC-MS .......................................................................... 36
2.7.2 Imaging-PAM measurements ............................................................................ 36
3. Results