Post-translational regulation and evolution of plant {γ-glutamate [gamma-glutamate] cysteine ligase [Elektronische Ressource] / presented by Roland Gromes

Rgromes

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

DISSERTATION

submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences















presented by

Diplom-Biologe Roland Gromes
born in: Heidelberg
Oral examination:







Post-translational regulation and
evolution of plant
γ-glutamate cysteine ligase




























Referees: Prof. Dr. Thomas Rausch
Prof. Dr. Rüdiger Hell Table of Contents
Table of Contents

1.1 Summary (English)..................................................................................................... 1
1.2 Zusammenfassung (Deutsch)............................................................................................ 2

2 Introduction................................................................................................................... 3
2.1 Glutathione: A central component of cellular sulfur metabolism ................................. 4
2.2 The establishment of glutathione homeostasis................................................................. 6
2.2.1 Glutamate cysteine ligase is the regulatory step of glutathione synthesis .................... 7
2.2.2 Plant glutamate cysteine ligase: Evolutionary relationship and subcellular localization
................................................................................................................................................ 9
2.2.3 Regulation of glutamate cysteine ligase activity......................................................... 11
2.2.3 Plant glutathione synthetase: Evolutionary relationship, subcellular localization and
regulation.............................................................................................................................. 13
2.2.4 Transport of glutathione in plants ............................................................................... 14
2.2.5 Degradation of glutathione in plants ........................................................................... 15
2.3 Stress and housekeeping metabolism – the multiple roles of glutathione................... 16
2.3.1 Glutathione as a redox metabolite 16
2.3.1.1 The basis of glutathione redox chemistry ............................................................ 16
2.3.1.2 The role of glutathione in the detoxification of reactive oxygen species (ROS) . 17
2.3.1.3 The role of glutathione in control of protein redox state...................................... 20
2.3.2 Involvement of glutathione in detoxification reactions............................................... 21
2.3.2.1 Glutathione S-transferases.................................................................................... 21
2.3.2.2 Glutathione and heavy metal tolerance ................................................................ 23
2.3.3 Glutathione as a regulator of gene expression, protein activity and development...... 25
2.3.3.1 Mechanisms of glutathione-dependent regulation of proteins and genes ............ 27

3 Results ............................................................................................................................. 30
3.1 The molecular mechanism for the redox regulation of Brassica juncea Glutamate
cysteine ligase (BjGCL) ......................................................................................................... 30
3.1.1 The crystal structure of the BjGCL protein shows two disulfide bridges................... 30
3.1.2 Knockout of the hairpin disulfide bridge (CC1) affects enzyme activity but not the K m
values of the substrates......................................................................................................... 32
3.1.3 The Core Disulfide Bridge CC2 of BjGCL mediates redox dependent dimer formation
.............................................................................................................................................. 34
3.2 The BjGCL mutant analogous to rml1 shows normal oligomerization behaviour but
is enzymatically inactive ........................................................................................................ 40
3.3 Sequencing, Cloning and Characterization of Nicotiana tabacum GCL..................... 41
3.4 Redox and GSH feedback regulation of plant GCL are mechanistically independent................... 46
3.5 Conservation of sequence motifs among plant and proteobacterial GCL proteins...49
3.5.1 The catalytic residues identified in BjGCL are highly conserved among plants and
proteobacteria ....................................................................................................................... 52
3.5.2 The residues involved in redox regulation of BjGCL are conserved only among plant
GCL sequences..................................................................................................................... 53
3.6 Cloning and characterization of proteobacterial GCL homologues ........................... 58
3.6.1 Proteobacterial GCL proteins are not inhibited by reduction and are functional as
monomers ............................................................................................................................. 62
3.6.2 Agrobacterium and Xanthomonas show an active glutathione metabolism ............... 65
- I - Table of Contents
3.7 The expression of plant GCL is affected by the availability of soluble thiols............. 67

4 Discussion...................................................................................................................... 71
4.1 The Crystal Structure of Brassica juncea GCL reveals unique features compared to
the Escherichia coli enzyme................................................................................................... 72
4.2 The redox regulation of BjGCL is dependent on two disulfide bridges...................... 75
4.2.1 Several lines of evidence point to a role of GCL redox regulation in vivo................. 79
4.4 Cysteine and glutathione regulate the activity of plant GCL via multiple mechanisms
.................................................................................................................................................. 81
4.5 The combination of redox and metabolite regulation allows an efficient control of
glutathione levels .................................................................................................................... 83
4.6 Proteobacterial glutathione biosynthesis is not subject to redox control.................... 86
4.7 The Evolution of Plant GCL can be traced by comparison of biochemical analysis
and in silico data..................................................................................................................... 88
4.7.1 Plants acquired their GCL genes via endosymbiosis or lateral gene transfer ............. 88
4.7.2 Redox regulation of plant GCL evolved in green algae, possibly in parallel to the
plastidic localization of the enzyme ..................................................................................... 90

5 Material and Methods........................................................................................... 93
5.1 Plant and Bacterial Culture ............................................................................................ 93
5.1.1 Plant material and Plant Cell Cultures ........................................................................ 93
5.1.2 Bacterial strains........................................................................................................... 93
5.1.2.1 Bacterial culture media and growth conditions........................................................ 94
5.1.2.1.1 List of Antibiotics used ......................................................................................... 94
5.1.2.1.2 Preparation of Glycerol Stocks ............................................................................. 95
5.1.2.2 Production of Competent Cells for Electroporation................................................. 95
5.1.2.3 Transformation of bacteria ....................................................................................... 95
5.2 Nucleic Acid Methods ...................................................................................................... 95
5.2.1 List of Plasmids........................................................................................................... 95
5.2.2 List of Oligonucleotides.............................................................................................. 96
5.2.3 DNA Methods ............................................................................................................. 97
5.2.3.1 Extraction of Genomic DNA from Bacteria ............................................................ 97
5.2.3.2 Extraction of Plasmid DNA from Bacterial Culture ................................................ 97
5.2.3.3 Determination of Nucleic Acid Concentrations....................................................... 97
5.2.3.4 Nucleic Aci