The Fourth Wave of Biocatalysis: Biochemical and in silico Characterization of FeS Cluster Containing Metalloenzyme Superfamilies 公开
Blue, Tamra (Summer 2023)
Abstract
The chemical manufacturing sector is one of the largest greenhouse gas and waste production zones in the United States. Enzyme biocatalysts have been at the forefront of industrial and pharmaceutical research as a green chemistry alternative to chemo-chemical methods. Enzymes are an attractive alternative as they have high stereospecificity and generate enantiomerically pure compounds without harsh solvents. However, a subset of enzymes has been traditionally underexplored, often due to their severe oxygen sensitivity. These metal cofactor-containing enzymes, or metalloenzymes, often have a propensity for radical initiation reactions catalyzing the stereoselective CH functionalization of unactivated carbons; however, they also display diverse reactivities.
This dissertation employs techniques honed in the most recent wave of biocatalysis innovation to unlock the untapped catalytic potential of iron-sulfur (FeS) metalloenzymes. These techniques highlight in silico approaches to enzymatic discovery and characterization. This includes genomics, bioinformatics, molecular docking, and molecular dynamic protocols coupled with traditional biochemical methods. The first chapter briefly introduces two metalloenzymes superfamilies of industrially interest and general in silico techniques.
The second chapter describes the implementation of a rationally designed protein library to probe the Old Yellow Enzyme (OYE) superfamily sequence space for novel enzyme candidates useful in the biosynthetic generation of monocarboxylic acid and decalone chiral building blocks. Additionally, the study assessed the standard operating procedure utilized to systematically explore the superfamily for novel activity. While this study yielded no monocarboxylic acid-compatible enzymes, four enzymes displayed activity for decalone chiral building block biosynthesis. Of those four, three were classified as insoluble under our standard operating conditions, indicating that the conditions are not universal.
The third chapter details the characterization of a metalloenzyme OYE subfamily with a unique mutation in its metal binding motif. This mutant displayed novel N-methyl-proline oxidative demethylation activity while being able to retain monocarboxylic acid reduction activity. The fourth chapter details our exploration of the ribosomally synthesized and post-translationally modified peptide (RiPP) recognition mechanism of radical SAM SuiB from Streptococcus suis. SuiB is a tailoring enzyme within a RiPP biosynthetic gene cluster of SuiA. However, it lacks interaction with the RiPP recognition domain. This chapter provides evidence for the non-conical identification of the precursor peptides by the tailoring enzyme’s bridging domain.
Table of Contents
Chapter 1. General Introduction. 1
1.1. General Introduction. 2
1.2. Metalloenzyme Biocatalysts. 4
1.3. Old Yellow Enzymes. 5
1.4. rSAM Enzymes. 9
1.5. Computational Sequence Analysis. 10
1.5.1.Sequence Similarity Networks. 10
1.5.2.Structure Prediction. 12
1.5.3.Molecular Docking. 12
1.5.4.Molecular Dynamics Simulations. 13
1.6. Heterologous Expression of Metalloenzymes. 14
1.7. Aims and Scope of the Dissertation. 15
1.8. Bibliography. 17
Chapter 2. Expanding the Industry Applications of the Old Yellow Enzyme Superfamily. 29
2.1. Introduction. 30
2.2. Results and Discussion. 36
2.2.1.OYE Protein Library Composition. 36
2.2.2.IVTT to efficiently produce protein. 36
2.2.3.Biochemical Evaluation of Protein Library. 37
2.2.4.Solubility characterization of the OYE Protein Library. 40
2.2.5.Solubility Relative to Activity and Species of Origin. 42
2.3.Conclusion. 43
2.4. Experimental 45
2.4.1.Materials. 45
2.4.2.Transformation and Protein overexpression. 45
2.4.3.Solubility Test. 46
2.4.4.IVTT of OYE Library Members. 46
2.4.5.Expression and purification of 3F3 (NemA) 47
2.4.6.Biotransformation. 48
2.4.7.Analytical methods. 48
2.5 Supplemental Information. 49
2.6. Bibliography. 55
Chapter 3. The OYE Superfamily Rides the 4th Wave of Biocatalysis 59
3.2. Results and Discussion. 62
3.2.1.Validation Of Longitudinal SSN.. 62
3.2.2.Bioinformatics Reveals Non-Canonical Fe/S-Cluster Binding Motif. 65
3.2.3.Expression Of Alanine Mutation Representative. 68
3.2.4.Optimization Of The Burkoye Activity For Library Screening. 70
3.2.5.Biochemical Evaluation Of Burkoye. 72
3.2.6.Validation Of [4Fe-4S] Cluster Incorporation. 80
3.2.7.Structural Prediction Of Burk OYE With Alphafold. 81
3.2.8.Genomic Neighborhood Analysis Of Burkoye. 83
3.3. Conclusion. 85
3.4. Experimental 86
3.4.1.Materials. 86
3.4.2.Local SSN generation. 87
3.4.3.Transformation, Expression, and Purification of BurkOYE.. 87
3.4.4.Anaerobic Expression. 88
3.4.5.Pseudo-Anaerobic Expression. 88
3.4.6.Aerobic Expression. 89
3.4.7.Purification. 89
3.4.8.pH Optimization. 90
3.4.9.Biotransformation. 91
3.4.10.Analytical methods. 91
3.4.11.BurkOYE Alphafold Prediction. 92
3.4.12.Genomic Neighborhood Analysis of BurkOYE.. 92
3.5. Supplemental Information. 92
3.6. Bibliography. 93
Chapter 4. In Silico Elucidation of RiPP Recognition in rSAM SuiB.. 101
4.1. Introduction. 102
4.2. Results and Discussion. 106
4.2.1.suiA Genomic Neighborhood Analysis. 106
4.2.2.Truncated SuiB RRE:SuiA-Fl fluorescence assay. 107
4.2.3.Characterization of RRE:SuiA interactions by ab initio docking. 108
4.2.4.Validation of docking approach for RiPP precursor and RRE.. 110
4.2.5.Capturing SuiB Protein dynamics via MD.. 111
4.2.6.Characterizing SuiA Secondary Structure. 113
4.2.7.Exploration of Disordered SuiA with SuiB.. 118
4.2.8.Hydrophobicity Analysis of SuiB and SuiA.. 121
4.2.9.Comparison to YydG from Bacillus subtilis. 126
4.3. Conclusion. 129
4.4. Experimental 131
4.4.1.SuiB:RRE-SuiA Fractional Saturation Assay. 131
4.4.2.suiA genomic analysis. 131
4.4.3.HADDOCK 2.2 ab initio Docking. 132
4.4.4.MD with GROMACS. 132
4.4.5.Structure and Simulation Analysis. 134
4.4.6.Hydrophobicity Analysis. 134
4.4.7.SuiA Conformation Change Analysis. 134
4.4.8.Circular Dichroism.. 135
4.5. Supporting Information. 135
4.6. Bibliography. 149
Chapter 5. Conclusion and Future Works. 158
5.1. General Conclusions. 159
5.2. Conclusions and Further Exploration of the OYE Superfamily. 159
5.3. Conclusions and Further Exploration of non-RRE mediated RiPP Recognition. 161
5.4. Bibliography. 163
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