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

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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 ........................................................................................ 81.1 Photosynthesis ........

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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 ............................................................................................... 37
3.1 Characterization of the enzyme encoded by 6A5t850 g0....................................... ..3..7 ....
V Table of contents
3.1.1 Identification of the candidate enzym...e. .......................................................... 37
3.1.2 Phylogenetic analysis............................................................................................................ 37
3.1.3 Cloning, heterologous expression and putrioifnic aof AtD-LDH ................................. .4.0
3.1.4 Quaternary structure of the enzyme................................................................ 40
3.1.5 Analysis of the prosthetic group ..................................................................... 41
3.1.6 Co-factor analysis and pH optimum ..................................................................................... 42
3.1.7 Substrate screening .......................................................................................... 43
3.1.8 Determination of kinetic constants ................................................................... 45
3.1.9 The catalytic site of AtD-LDH ............................................................................ 46
3.1.10 Isolation of knock-out mutants .......................................................................................... 46
3.1.11 In-gel assays of knock-out plant.s. ................................................................ 47
3.1.12 Plant feeding experiments ........................................................................... 48
3.2 Characterization of the enzyme encoded by 6A4t040 g3...................................... .5..1 .....
3.2.1 Identification of the candidate enzym...e. ........................................................................... 51
3.2.2 Phylogenetic analysis........................................................................................ 51
3.2.3 Cloning, heterologous expression and putrioifnic aof AtD-2HGDH ............................ ..5..4.
3.2.4 Quaternary structure of AtD-2HGDH ................................................................. 54
3.2.5 Analysis of the prosthetic group .......................................................................................... 55
3.2.6 Co-factor analysis and pH optimum ............................................................... 56
3.2.7 Substrate screening .......................................................................................... 57
3.2.8 Determination of kinetic constants ................................................................... 57
3.2.9 The catalytic site of AtD-2HGDH .......................................................................................... 59
3.2.10 Isolation of knock-out mutants ................................................................... 59
3.2.11 In-gel assays of knock-out plant.s. ................................................................ 60
3.2.12I n silico co-expression analysis ..................................................................... 62
3.2.13 Dark-induced senescence and metabolitel ipnrg o.f..i........................................................ 63
3.3 Characterization of the enzyme encoded by 8A36t40 g1...................................... .6..7 .....
3.3.1 Identification of the candidate ...................................................................... 68
3.3.2 Isolation of knock-out mutants .................................................................... 70
3.3.3 In-gel assays of knock-out mutant.s. .................................................................................... 70
3.3.3 Cloning, heterologous expression and putrioifnic aof AtGOX3................................... ..71
3.3.4 Co-factor analysis and pH optimum ............................................................... 71
3.3.5 Substrate screening .......................................................................................... 73
3.3.6 Determination of kinetic constants ...................................................................................... 73
3.3.7 Structural considerations ................................................................................ 74
VI Table of contents
3.3.8 Plant feeding experiments .............................................................................. 74
4. Discussion .......................................................................................... 76
4.1 At5g06580 encodes a D-LDH ......................................................................... 76
4.1.1 AtD-LDH is conserved in evolution and elso ctaol imz itochondria ............................. .7..6
4.1.2 AtD-LDH is specific for D-lactate and .D.-..2.H..B. ................................................................ 77
4.1.3 AtD-LDH metabolizes D-lactiant ev ivo ..................................................................... 77
4.1.4 Plants may have a second MG detoxificathiwona y pa............................................. 78
4.1.5 Model ............................................................................................................. 79
4.1.6 Outlook ........................................................................................................... 80
4.2 At4g36400 encodes a D-2HGDH ..................................................................................... 80
4.2.1 AtD-2HGDH is conserved in evolution andi zelso ctaol mitochondria ........................ ..8.0. ..
4.2.2 D-2HG accumulates di2nh gdh mutants..................................................................... 81
4.2.3 D-2HG is most likely produced in a coonnd ernesactition ....................................... 82
4.2.4 Model ................................................................................................................................... 82
4.2.5 Outlook ............................................................................................................ 83
4.3 At4g18360 encodes an (S)-2-hydroxy-acid o x.id..a..s.e...................................... .8.4 ....
4.3.1 AtGOX3 oxidizes glycolate and L-lactateh ighw iethf ficiency ..................................... 84
4.3.2 Identifying important amino acids in thley ticca tsaite ........................................................ 84
4.3.3I n vivo role of AtGOX3 ................................................................................... 85
4.3.4 Outlook ........................................................................................................... 85
5 Conclusions and future directions ..............................................8.6.. ......
6. References ......................................................................................... 88
7. Abstract ............................................................................................. 99
8. Kurzzusammenfassung .............................................................. ..1.0.0. .....
9. Acknowledgements ............................................................................ ..1.0.1. ...
10. Erklärung ......................................................................................... 1 02

VII Introduction
1. Introduction

1.1 Photosynthesis
Photosynthesis is a process of crucial importoarn cle iffe on earth. It is the process by which
some photoautotrophic organisms capture light eneyr gfrom the sun and use it to fix carbon
dioxide from the atmosphere. As a by-products opfr otcheiss oxygen is produced. Almost all
hydrocarbons found in nature have once existedre ea s cafrbon dioxide and subsequently
been fixed by photosynthetic organisms. Consequey,nt lthese organisms not only produce
the food we eat, but also the oxygen we breoatohes.y nPthhesis is generally divided up in
two parts, the light reactions and the “dark”i ornesa. ct

1.1.1 Light reactions
Oxygenic photosynthesis performed by plants, alngad asome photosynthetic bacteria uses
two photosystems working in sequence. The proceaskse st place in the thylakoid membrane
of the chloroplasts where the photosynthetic lrieghatc tions absorb light from the sun and
capture it in the form if chemical bonds. T hiiss fpeearftormed by photosystem I (PSI) and
photosystem II (PII) in conjunction with etchteiivre relsigpht harvesting complexes. Photons
captured by the chlorophylls in the light-harvg esctoinmplexes are transferred to special
protein- chlorophyll reaction centers inside ehoatcoh sypstem through excitation transfer. In
the reaction center an electron from one of otrhoep hyclhls is excited and subsequently
transferred to a series of acceptors in the ne letrcatnrosport chain. The linear electron
transport chain builds up a proton gradient oeve rm ethmbrane, which is used to convert
+ADP to ATP, and reduces N ADtoP NADPH. The light reactions thus ultimatelye s setrov
produce reduced energy carriers which are furtheser d uin the dark reactions to fix
atmospheric CO (Taiz and Zeiger, 2006). 2

1.1.2 The dark reactions
The NADPH and ATP originating from the light reoancst iare used in the Calvin-Benson cycle
to fix atmospheric carbon. In a carboxylatioino n reraibcutlose-1,5-bisphosphate (RuBP) – a
5-carbon unit – is converted into two molecu3l-epsh osopfh oglycerate – a 3-carbon unit.
The 3-phosphoglycerate is further reduced to 2s,p3ho-bspihoglycerate and finally
glyceraldeyde-3-phosphate. Glyceraldehyde-3-phostpeh a exists in equilibrium with
dihydroxyacetone phosphate. These photoassimilatesc an further be exported to the cytosol
by the triose phosphate/phosphate translocator (ggFel,ü 1999), be condensed into fructose-
6-phosphate and enter starch synthesis, or re-e ntther Calvin-Benson cycle to form RuBP.
thTypically 1/6 of the generated glyceraldehyde-3-phosphate iso retexdp or used for starch
thsynthesis while 5/6 re-enters the Calvin-Benson cycle. The key enizny mtehi s cycle,
catalyzing the carboxylation reaction, is ribu1l,o5-seb-isphosphate carboxylase/oxygenase
(RuBisCO). This is a hexadecameric enzyme in hig heprlants, consisting of eight identical
large subunits, encoded in the chloroplast, hat ndi eenitgical small subunits, encoded in the
nucleus (Miziorko and Lorimer, 1983; Parry 2et00 3a)l. .,D espite the supreme importance of
8 Introduction
this enzyme for all land plants it is notnoerifofuisclieyn t,i catalyzing only about 4
carboxylation reactions per second (Tcherkez ,e t 2a00l6.). Plants have adapted to this by
producing large amounts of the enzyme and RuBisaCnO cmake up as much as 30-50% of
total leaf protein in C3 plants (Parry et) . al ., 2003
1.2 Photorespiration
In addition to being an extremely slow enzymseC,O cRaunBniot properly discriminate
between oxygen and carbon dioxide. Consequentley ,e ntzhyme does not only catalyze the
carboxylation of RuBP, but also the oxygenatioen . laTthter reaction produces one 3-
phosphoglycerate as well as the 2-carbon unit sp2h-opglhoycolate (Lorimer, 1981; Ogren,
2003). This 2-phosphoglycolate cannot be readileyd usby the cell and must first be
converted back to the Calvin-Benson cycle inteartme ed3i-phosphoglycerate through the
photorespiratory pathway, stretching over chloropstlsa, peroxisomes, mitochondria and
the cytosol (Maurino and Peterhänsel, 2010; To,l b1e99r7t; Wingler et al., 2000) (Fig. 1). The
pathway is ubiquitous in photosynthesizing organisms, being present in plants (Maurino and
Peterhänsel, 2010; Tolbert, 1997), unicelluel a(r Saulzugaki et al., 1990; Tian et al., 2006) and
cyanobacteria (Bauwe, 2010; Eisenhut et al., 2 008).

1.2.1 The photorespiratory pathway
The importance of the photorespiratory pathway insd eurscored by the fact that most
mutants lacking activity in any of the particg ipeantiznymes are lethal or display severely
compromised growth at ambient C O levels. They can, however, be rescued at high CO2 2
levels where no photorespiration occurs. Photoraetsiopni comprises a long series of
enzymatic reactions (Fig. 1).


9
Introduction

Figure 1. The photorespiratory pathw ayT.he photorespiratory pathway serves to convert 2-
phosphoglycolate back to 3-phosphoglycerate. Phoetsopriration derives its name from the fact
that it is a light-dependent uptake of oxygen BbisyC O Rwuith subsequent carbon dioxide release.
The figure is re-produced from Maurino and Petserlhä n(2010).

Upon oxygenation of RuBP and subsequent formatfio n2 -ophosphoglycolate this molecule is
dephosphorylated into glycolate by 2-phosphoglytceo laphosphatase (PGLP) inside the
chloroplast (Schwarte and Bauwe, 2007; Somernvidl leOg rean, 1979). Glycolate is further
exported to peroxisomes where it is oxidized otoxy lglayte by glycolate oxidase (GO), with
the production of OH (Xu et al., 2009; Zelitch et al.A,r ab2i0d09o)p.s is thaliana contains 2 2
three GOs that are closely related to each otOhXer1 ,( GGOX2 and GOX3) and two GO-like
proteins (HAOX1 and HAOX2) (Reumann et al., 2F0ig0.4 )2 )(. There are differences in
sequence as well as differences in which tisseu eg enthes are expressed, as determined by
microarray experiments (http://bar.utoronto.ca/ecfgip-b/in/efpWeb.cgi) (Kamada et al.,
2003; Winter et al., 2007). At3g14420 (AtGOX1) Aat3ngd1 4415 (AtGOX2) are both highly
expressed in plant leaves and most likely tpea ritnic ippahotorespiration (Xu et al., 2009). In
contrast, At4g18360 (AtGOX3) is expressed at mouwche r llevels and almost exclusively in
roots. At3g14130 (AtHAOX1) and At3g14150 (AtHAOXar2e) expressed at intermediate levels
and exclusively in developing seeds. The physciaol ofgiunction of these latter three enzymes
is currently unknown.

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