Generating designer enzymes for therapeutic and industrial applications Pubblico
Daugherty, Ashley Bagwell (2013)
Published
Abstract
Enzymes are versatile catalysts that are important for their use in a variety of therapeutic and industrial processes. Often times enzyme's inherent properties are not suitable for a particular application and require engineering to improve substrate specificity, stability, reactivity, enantioselectivity etc. In this dissertation semi-rational design strategies in combination with circular permuation were employed to alter the biocatalytic properties of three separate enzyme classes toward their respective applications. In each case, the engineering methods resulted in dramatically reduced mutant library size and variants were generated with enhanced designer properties.
Structural guided rational design and computational tools were used to reengineer the active site of deoxyribonucleoside kinases dCK and DmdNK to have specificity toward 3'-modified nucleoside analogs ddT and kT over the natural substrate thymidine. This method proved successful for quickly tuning substrate specificity towards nucleoside analogs using smaller focused libraries.
Secondly, Rosetta fixed backbone design was employed to redesign subtilisin BPN' propeptide, as structural stabilization has been proposed to competitively inhibit protease self-cleavage. Computationally optimized propeptide sequences were generated with increased structural integrity and enhanced thermostability. Experimental characterization verified their structural stabilization and enhanced thermostability but inhibition profiles suggest that propeptide stability alone is insufficient for effective inhibitor design. Overall, this approach offers a convenient highly tunable method for the design of future protease inhibitors.
Thirdly, a completely synthetic circular permutation library of Old Yellow Enzyme from Saccharomyces pastorianus (OYE1) was created using a whole gene synthesis method. Subsequently, a cell-free in vitro transcription/translation screen was employed to assess the impact of termini relocation throughout the protein sequence against substrates: ketoisophorone, cinnamaldehyde and (S)-carvone. Library screening identified over 70 variants with enhanced catalytic activity and several showed over an order of magnitude improvement. Interestingly, location of new termini in all improved variants were in the same four loop/lid regions near the active site. Characterization revealed identical secondary structures, altered oliogmeric states, and increased thermostability between the permutants studied. The crystal structure of a top variant, cpOYE303, was solved and verified a significantly more open and accessible active site with altered flexibility near locations of new and old termini.
Table of Contents
Table of Contents
Chapter 1: General Introduction...1
1.1 General Introduction...2
1.1.1 General applications of enzymes...2
1.1.2 Protein engineering approaches...3
1.2 Deoxyribonucleoside kinases in nucleoside analog prodrug activation...9
1.2.1 Nucleoside analogs as anti viral and anti
cancer therapies...9
1.2.2 dNK family...13
1.2.4 Limitations with endogenous dNKs...14
1.3 Subtillisin proteases and their propeptide
component...17
1.4 Enoate reductases in biocatalysis...22
1.4.1 Old Yellow Enzyme family...23
1.4.2 Structural features and catalytic mechanism of OYE...24
1.4.3 OYE engineering...26
1.5 Aim and scope of the dissertation...29
1.6 References...31
Chapter 2: Engineering Deoxyribonuceloside Kinases for 3'-Modified Nucleoside Analogs...39
2.2. Results and discussion...44
2.2.1. Transfer of beneficial mutations between
DmdNK and hdCK...44
2.2.3 Rosetta designer kinase for kT...51
2.2.4 Refinement of kT designer kinase...56
2.2.5. In vitro characterization of final kT Mutants...57
2.3. Conclusion remarks...60
2.4. Materials and methods...61
2.4.1 Materials...61
2.4.2 Site-directed mutagenesis dCK mutants...62
2.4.3 Site-Directed Mutagenesis DmdNK mutants...62
2.4.4 Protein overexpression in E. coli BL21(DE3)pLysS...63
2.4.5 His-tagged Protein Purification...64
2.4.6 Protein overexpression in E. coli K12 TB1...65
2.4.7 MBP-tagged protein purification...65
2.4.8 Kinetics...66
2.4.9 CD Spectroscopy...66
Chapter 3: Novel Protease Inhibitors via Computational Redesign of Subtilisin BPN' Propeptide...71
3.1 Introduction...72
3.2 Results and Discussion...73
3.2.1 Computational propeptide
redesign...73
3.2.2 Expression and purification of designer
propeptides...84
3.3.3 Structural characterization of propeptides...87
3.3.4 Functional characterization of propeptides...92
3.3 Conclusions...95
3.4 Material and Methods...97
3.4.1 Materials...97
3.4.2 Computational simulations...97
3.4.3 Molecular Dynamics...98
3.5.6 Gene synthesis...99
3.4.7 Site-directed mutagenesis...100
3.4.8 Protein expression and purification in the IMPACT
system...100
3.4.9 Tag-less protein expression...101
3.4.10 Tag-less protein purification...102
3.4.11 Circular dichroism spectroscopy...103
3.4.12 Intrinsic tryptophan fluorescence...103
3.4.13 Nuclear magnetic resonance spectroscopy...104
3.4.14 Protease inhibition...104
Chapter 4: Circular Permutation of Old Yellow Enzyme: Characterization of a Complete Synthetic Library...109
4.1 Introduction...110
4.2 Results and Discussion...115
4.2.1 OYE1 library generation...115
4.2.2 Development of a high throughput IVTT screen...117
4.2.3 Library Screening-KIP...120
4.2.4 Detailed characterization of cpOYE variants...123
4.2.5 Exploring other substrates: cinnamaldehyde and
S-carvone...125
4.2.6 Rapid reaction kinetics...128
4.2.7 p-Hydroxybenzaldehyde Binding Studies...131
4.2.8 Secondary Engineering Cofactor Analogs...134
4.3 Conclusions...142
4.4 Materials and Methods...145
4.4.1 Materials...145
4.4.2 cpOYE1 library synthesis...145
4.4.3 Primary library screening...146
4.4.4 Protein expression and purification...147
4.4.5 Spectral properties of OYE1 variants...148
4.4.6 Activity assays...149
4.4.7 Stopped flow experiments...150
4.4.8 Cofactor analog incorporation...151
4.4.9 Desaturase activity assays...151
4.5 References...153
Chapter 5: Structural Exploration of Representative OYE Permutants...161
5.1 Introduction...162
5.2 Results and discussion...165
5.2.1 Investigation of secondary structure and
thermal denaturation of permutants...165
5.2.2 Oligomeric state as a result of permutation...168
5.2.3 Crystallographic analysis of cpOYE variants...170
5.2.4 cpOYE303-full length crystal structure...173
5.2.5 Creation of truncation mutant and activity
analysis...174
5.2.6 Crystallization of truncated cpOYE303...175
5.2.7 cpOYE303-truncated crystal structure...177
5.2.8 Comparison of cpOYE303 crystal structures with
wt-OYE...177
5.3 Conclusions...183
5.4 Materials and Methods...183
5.4.1 Circular Dichroism
Spectroscopy...183
5.4.2 Determination of Oligomeric State...184
5.4.3 Protein Expression and Purification for
Crystallization...184
5.4.4 cpOYE303 Crystallization...184
5.4.5 Crystal data collection...185
5.4.6 Construction of cpOYEG303 truncation...185
5.4.7 Construction of cpOYE-W116I mutants...186
5.4.8 Kinetic analysis of variants...187
5.5 References...189
Chapter 6: Conclusions and Perspectives...193
6.1 Summary...194
6.2 A starting point for evolving future 3' modified nucleoside
analog kinases...194
6.3 Future investigation of subtilisin BPN' propeptide...195
6.4 Continuing to teach Old Yellow 'new tricks'...196
6.5 References...200
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