Expression and Functional Characterization of Monoamine Oxidase from the Zebrafish (Danio Rerio): Comparisons with Human Monoamine Oxidases A and B Open Access

Kacar, Betul (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/2801ph06z?locale=en
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Abstract

Monoamine Oxidases (MAO) are flavin containing enzymes located in the outer mitochondrial membrane. Mammals, including humans are shown to contain two forms of this enzyme as MAO A and MAO B. However, not all organisms contain two separate genes expressing these enzymes. Recent studies have shown that zebrafish, a popular teleost organism suitable for various pharmacological applications, contains a single MAO gene. It was proposed that human and teleost MAOs are co-orthologs and share a single common ancestor that underwent a gene duplication event. In addition, studies with whole zebrafish neural tissues have shown that zebrafish MAO exhibit properties closer to human MAO A. To test this hypothesis and to provide the first detailed characterization of zebrafish MAO (zMAO), we developed a high-level expression and purification system for zMAO where we could obtain 235 mg of protein from 0.5 L culture of Pichia pastoris. Then we performed the first detailed functional analysis of the protein with various MAO A and MAO B specific substrates and inhibitors. Here, we also present a comprehensive analysis of quantitive structure relationship of zMAO catalysis in comparison with the human MAO isoforms. Overall data suggest that zMAO contains the properties of both human MAO A and MAO B with properties closer to those of MAO A. The studies from this dissertation provide extensive analysis of this single form of the enzyme and are aimed to be helpful in the pharmacological studies that target designing better drugs targeting MAO using this zebrafish as a system.

Table of Contents

Table of Contents
CHAPTER 1 - Introduction to Monoamine Oxidases
PART I: Human MAO A and MAO B...1

1.1 Pharmacological Importance of MAO...1

1.1.1 Molecular Characteristics of Human MAO A and Human MAO B...2
1.1.2 Structural Analysis of Human MAO Isoforms...4

1.1.2.1 Flavin Binding Domain...10
1.1.2.2 Substrate Binding Domain...15
1.1.2.3 Membrane Binding Domain...19

1.1.3 MAO Substrates and the Amine Mechanism...21
1.1.4 Properties and Importance of MAO Inhibitors...26

1.1.4.1 Irreversible Inhibitors...27
1.1.4.2 Reversible Inhibitors...29

PART II: Zebrafish MAO...34

1.2 What makes Zebrafish MAO important?...34

1.2.1 Molecular Properties...34
1.2.2 Comparison to Human MAO A and MAO B...37
1.2.3 Importance of Zebrafish in Drug Development...43

1.3 Dissertation Objectives...44
1.4 References...46

CHAPTER 2 - High-level expression and purification of zebrafish monoamine oxidase in Pichia pastoris

2.1 Introduction...54
2.2 Materials and Methods...55

2.2.1 Materials...55
2.2.2 Cloning of zebrafish MAO...56
2.2.3 Transformation of zebrafish MAO gene into P. Pastoris...57
2.2.4 Expression of zebrafish MAO...57
2.2.5 Purification of zebrafish MAO...58
2.2.6 Determination of the Protein Content...61
2.2.7 Steady-State Kinetics...61
2.2.8 Mass Spectroscopy...62
2.2.9 HPLC Separation...63
2.2.10 Fluorescence Studies...64
2.2.11 Thermal Stability...64
2.2.12 Oxygen Affinity...65
2.2.13 Data Analysis...65

2.3 Results...65

2.3.1 Enzyme expression and purification...65
2.3.2 Characterization of Zebrafish MAO...67
2.3.3 Spectral Properties...68
2.3.4 Determination of the nature of the covalent linkage...71
2.3.5 Identification of the Flavin...75
2.3.6 Protein Sequence Analysis...76
2.3.7 Thermal Stability...78
2.3.8 Catalytic Properties...80

2.4 Discussion...83
2.5 References...87

CHAPTER 3 - Functional Analysis of Zebrafish MAO...91

3.1 Introduction...91
3.2 Materials and Methods...91

3.2.1 Materials...91
3.2.2 Preparation of Enzyme...92
3.2.3 Steady-state Kinetics Experiments...93
3.2.4 Competitive Inhibition...95
3.2.5 Data Analysis...95
3.2.6 Enzyme Functionality...95

3.3 Results...96

3.3.1 Steady-State Kinetic Properties for the zMAO...96
3.3.2 Competitive Inhibition...98

3.3 Summary...107
3.4 References...112

CHAPTER 4 - Comparative Structure-Activity Relationships of Zebrafish Monoamine Oxidase

4.1 Introduction...116
4.2 Materials & Methods...119

4.2.1 Materials...119
4.2.2 Preparation of the enzymes...120
4.2.3 Determination of kinetic parameters: kcat, Km and Kd...120
4.2.4 Steady state kinetic measurements of para- and meta- substituted benzylamine analogue oxidation...121
4.2.5 Steady state kinetic measurements of para- and meta- substituted phenylethylamine analogue oxidation...123
4.2.6 Data analysis...123

4.3 Results...124

4.3.1 Steady state kinetic measurements with para-substituted benzylamine analogues and the effects of isotopic substitution...124
4.3.5 Steady state kinetic measurements with meta-substituted benzylamine analogues and the effects of isotopic substitution...126
4.3.7 Steady state kinetic measurements of para-substituted phenylethylamine analogues...129
4.3.9 Steady state kinetic measurements with meta-substituted phenylethylamine analogues...132

4.3. Quantitive Structural Activity Relationships Describing zMAO Catalysis...134

4.3.1 QSAR in para- and meta- substituted benzylamine analogues binding to zMAO...138
4.3.2 QSAR in meta- and para- substituted phenylethylamine analogues binding to zMAO...144

4.4 Discussion...156

4.4.1 Overview of the QSAR Data...156
4.4.1 Mechanistic and Structural Interpretation...161

4.5 References...167

Chapter 5 - Conclusion & Future Work...170

5.1 Summary of the Results...170
5.2 Reflections and Future Work...174
5.3 References...177

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