Exploring the Role of Cofactor and Protein Sequence Variability Across the Old Yellow Enzyme Superfamily Pubblico

Abstract

Over the past century enzymes have been the center of intense research efforts focused on the discovery and engineering of novel biocatalysts to produce industrially and commercially relevant chemical compounds. Most of these efforts involve enhancing enzyme efficiency, stereospecificity, stability, and substrate scope through alterations to the native protein backbone. Certain enzymes, however, require small molecule chemical cofactors to perform their native functions. Accordingly, engineering of natural cofactors represents parallel strategy for enzyme customization that can be used alongside traditional backbone modification methodologies. On a more fundamental level, cofactor engineering may also be employed to probe the mechanistic and biophysical characteristics of protein catalysts.

This dissertation thus utilizes engineered cofactor analogs to examine the functional role of the native cofactor flavin mononucleotide (FMN) in the folding of Old Yellow Enzyme 1 (OYE1) from Saccharomyces pastorianus. Preliminary work with a cell-free protein expression system determined that FMN must be present during OYE1 synthesis to produce catalytically functional enzymes, disputing a longstanding assumption that the cofactor’s presence is unnecessary for proper folding. Nascent OYE1 was then supplemented with cofactor analogs containing key elements of the native FMN in order to systematically study the contribution of specific chemical groups towards proper enzyme folding. Activity assay data suggest that the flavin’s isoalloxazine and terminal phosphate moieties are essential for productive OYE1 folding.

With the endless industrial demand for new biocatalysts, this work also details our exploration into the OYE superfamily in search of novel enzymes. Using sequence similarity networks (SSNs) to visually guide our sampling efforts, we have doubled the number of characterized OYEs and have begun functionally profiling the entire superfamily in an unprecedented manner. Excitingly, we have identified a number of novel catalysts capable of catalytically out-performing any of the previously reported native or engineered OYE family members.

Table of Contents

Chapter 1: General Introduction 1

General Introduction 2

Chemical Versatility of Cofactors 3

Artificial Metalloenzymes (ArMs) 11

Flavin Cofactors 15

Flavoenzymes 18

Old Yellow Enzymes 24

Beyond OYE1, the OYE superfamily 28

Cell-Free Protein Expression 31

Aims and Scope of the Dissertation 33

References 34

Chapter 2: Truncated FAD synthetase for direct biocatalytic conversion of riboflavin and analogs to their corresponding flavin mononucleotides 54

Abstract 55

Introduction 55

Results and discussion 59

Heterologous expression and purification of full-length and truncated FADSs 59

Characterization of secondary structure and stability by circular dichroism spectroscopy 61

Catalytic activity with RF and flavin analogs 62

Supplemental Information 65

Materials and Methods 65

References 71

Chapter 3: Elucidating the Role of FMN in the Productive Folding of Old Yellow Enzyme 1 73

Introduction 74

Synthesis of FMN Analogs 78

Cofactor Analogs 1, 3, 4 78

Cofactor Analog 2 79

Results and Discussion 81

ITC Binding Studies of FMN (analogs) 81

PURE System Folding Studies of OYE1 84

Conclusion 87

Experimental 89

General Information 89

Synthesis of (2R,3S,4S)-5-(2,6-dioxo-1,2,3,6-tetrahydro-9H-purin-9-yl)-2,3,4-trihydroxypentyl phosphate (1) 89

Synthesis of (2R,3S,4S)-5-(9H-carbazol-9-yl)pentane-1,2,3,4-tetraol (2) 91

Synthesis of (2R,3S,4S)-5-ammonio-2,3,4-trihydroxypentyl phosphate (3) 93

Synthesis of (2R,3S,4S)-2,3,4,5-tetrahydroxypentyl phosphate (4) 93

Conversion of Deoxyriboflavin to DeoxyFMN 94

Expression and Purification of OYE1 94

Isothermal Calorimetry Binding Studies 96

PURExpress In vitro Transcription/Translation of OYE1 with FMN (analogs) 96

Cofactor Competition and Enzymatic Activity Assay 97

References 98

Chapter 4: Exploration of the Old Yellow Enzyme Superfamily Utilizing Sequence Similarity Networks 100

Introduction 101

Results and Discussion 107

Sequence Similarity Network (SSN) 107

Selection of Novel OYE Family Members 110

Evaluation of Novel OYE Family Members 110

Solubility of Novel OYE Family Members 118

Conclusion 121

Experimental 123

General Information 123

PURExpress In vitro Transcription/Translation of OYE Library Members 123

Enzymatic Activity Assay for OYE Library Members 124

Solubility of OYE Library Members 125

Supplemental 126

References 133

Chapter 5: Conclusions and Future Work 143

General Conclusions 144

Continued Use of Cofactor Engineering 145

Further Exploration of the OYE Superfamily 147

References 151

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry
Parola chiave	OYE FMN cofactor engineering
Committee Chair / Thesis Advisor	Simon Blakey, Emory University Stefan Lutz, Emory University Emily Weinert, Emory University

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