Enzymes catalyze specific reactions in almost all cellular processes, including DNA replication and repair, transcription, translation, signal transduction and energy production. Therefore, extensive efforts are underway to understand the functions and mechanisms of these processes. The potential contribution of the conformational dynamics of enzymes to their high catalytic power has received particular attention in the last decades. Studies indicate that protein dynamics are involved in substrate binding and product release; however, the role of dynamics in catalysis is still controversial. Here, we investigate the substrate-dependent dynamic properties of the Escherichia coli AlkB protein, and the role of a specific dynamic transition in the efficiency of the catalytic reaction cycle. AlkB is an iron/2-oxoglutarate (Fe(II)/2OG) dependent dioxygenase, which removes certain cytotoxic alkyl lesions from DNA and RNA bases that are not repaired by other known mechanisms. Using Fe(II) as a cofactor and 2OG and molecular oxygen as co-substrates, AlkB catalyzes a multistep redox reaction in which first, 2OG is oxidized yielding succinate, carbon dioxide and a reactive oxyferryl (Fe(IV)=O) intermediate; second, the alkylated base is hydroxylated by the Fe(IV)=O intermediate, and third, the hydroxylated base spontaneously resolves upon release from the enzyme. Our fluorescence and NMR spectroscopic data demonstrate that a microsecond-tomillisecond timescale conformational transition in the nucleotide recognition lid (NRL) of AlkB regulates the correct sequential order of substrate binding, i.e. Fe(II) and 2OG first, followed by the DNA substrate. By combining isothermal titration calorimetry with NMR, we show that less than 20% of the residues in AlkB become ordered during this conformational transition, indicating that this conformational change is mostly localized to the NRL, while the conformation of the dioxygenase core is minimally altered. In mutant AlkB variants that perturb the dynamics of this transition, 2OG is oxidized generating the Fe(IV)=O intermediate; however, the reaction cycle cannot be completed due to the premature release of the alkylated DNA substrate, leading to uncoupled turnover of 2OG. These data demonstrate that the conformational dynamics control the catalytic efficiency of AlkB. Our results further extend the view on the role of protein dynamics in substrate binding or product release by emphasizing the importance of protein dynamics for coupling sequential sub-reactions in a complex multistep reaction cycle. This finding illustrates a striking example of the relation between protein dynamics and overall enzyme efficiency.

As the demand for cost-efficient and environmentally friendly processes increases in the chemical industry, impact of biocatalysis, which is the utilization of enzymes and whole microorganisms for production of fine chemicals, has become more predominant. From pharmaceuticals to cosmetics, biocatalysts are widely used in various sectors, and their significance have dramatically intensified with the introduction of initial protein engineering techniques in 1980s. As the field of protein engineering has evolved over the last few decades, its integration with other disciplines such as process engineering and synthetic biology is now more critical for establishing non-natural pathways and reactions to produce broader range of chemicals. While developing an interdisciplinary approach, few strategies have emerged to be more prevalent: (i) better integration of biocatalysts with (nano)devices, and (ii) use of protein based scaffolds for creating novel synthetic multienzyme cascades. Throughout this doctoral thesis, applicability of these ideas with oxidoreductases was investigated. Oxidoreductases are a class of under-utilized enzymes that catalyze the electron transfer between different metabolites, while at the same time use cofactors (NAD(P)(H), molecular oxygen, etc.) as the electron supplier. In Chapter 2, the electron transfer mechanism of a monooxygenase, cytochrome P450 27B1 (CYP27B1), was mimicked for electrochemical sensing of a vitamin D form (25(OH)D) in solution. Natural electron transfer pathway of this enzyme uses NADPH and two electron transfer proteins for conversion of 25(OH)D to its product. Inspired by this mechanism, this enzyme was mixed with an artificial redox mediator and immobilized on an electrode surface. As a result of rigorous experiments, CYP27B1-modified electrode was found to detect 25(OH)D in its physiological range. This is a significant result as it opens a new way for development of a vitamin D biosensor that can diminish the amount of required cost and time for testing. In the next chapter of the thesis, effects of changing the size of cofactor on catalysis of dehydrogenases were studied in detail. Natural cofactors of two different redox enzymes were chemically modified with PEG, and kinetic experiments were conducted in order to better understand the relation between transport phenomena and biocatalysis. It was found that when the size of the cofactor was increased, two enzymes were affected differently; while efficiency of one enzyme was not altered significantly, that of the other dropped dramatically. Through comprehensive analysis, dominant impact of PEGylation was determined to be due to the differences in the interactions of PEGylated cofactors and enzymes. This study showed that protein engineering methods can be utilized to gain insights into better understanding of the relationship between mass transfer and catalysis in engineered bioprocesses and biocatalytic cascades. In Chapter 4, PEGylated cofactors were used to create artificial multienzyme complexes. In this study, SpyCatcher-SpyTag scaffold was utilized for wiring two redox enzymes and by tethering with PEGylated cofactors, a new biocatalyst with self-contained redox chemistry was obtained. Detailed kinetic analysis showed that this new multienzyme cascade was able to catalyze a reaction that was thermodynamically downhill but kinetically very slow in the absence of any enzyme. This also proved that attached cofactor acts as a ‘swing-arm’, carrying electrons from one enzyme to another; similar to the unique mechanism of pyruvate dehydrogenase complex. Generality of this methodology was investigated by constructing an immobilized three-enzyme-containing biocatalyst, which was hypothesized to catalyze an industrially important reaction under very mild conditions. This work is a significant contribution to the field, and a good demonstration of use of protein engineering for process engineering applications. Chapter 5 concludes this thesis with a study that investigates the practicability of a collagen mimetic peptide as a novel way of constructing multiprotein cascades. Collagen mimetic peptides are composed of three individual strands that might (homotrimer) or might not (heterotrimer) have identical sequences, and in this work, we have utilized a recently designed hydroxyproline-free sequences of a heterotrimer collagen mimetic peptide. Individual strands were attached to different proteins by genetic fusion, and optimum experimental conditions for self-assembly of a multiprotein complex were investigated. Initial results suggested formation of such a complex, but further experiments are required to finalize the confirmation. This new collagen-based platform studied in this chapter is a crucial step towards development of cofactorless multienzyme cascades. Finally, this doctoral thesis demonstrates the prominence of protein engineering in biocatalysis applications by utilizing various strategies together with the electron transfer mechanisms of oxidoreductases. By expanding and building upon these methodologies, it is possible to obtain more improved biosensors and functional artificial multienzyme cascades with industrial applications. Hence, this study is a promising example to exhibit the impact of interdisciplinary approach on industrial biotechnology.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 160-170).

Dissertation (Ph.D.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 196-212).

by Fong Wing-ping. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1987. / Bibliography: leaves 186-198.

Thesis (M. S.)--Michigan State University. Dept. of Food Science and Human Nutrition, 2006. / Title from PDF t.p. (viewed on June 19, 2009) Includes bibliographical references (p. 67-70). Also issued in print.

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 86-100). Also available on the World Wide Web.

Chong, King Wai Isaac. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 99-104). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Table of Contents --- p.vi / Lists of Figures --- p.ix / List of Tables --- p.xi / List of Abbreviations --- p.xii / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1.1 --- Overview of Glyceraldehyde 3-phosphate Dehydrogenases --- p.1 / Chapter 1.2 --- Properties And Molecular Structures of GAPDH --- p.3 / Chapter 1.3 --- Action Mechanism of GAPDH --- p.6 / Chapter 1.4 --- Novel Functions of GAPDH Unrelated to Carbohydrate Metabolism --- p.8 / Chapter 1.5 --- Effects of Heavy Metal on Enzyme Activity And Gene Expression of GAPDH --- p.10 / Chapter 1.6 --- Metal Binding Properties And Metal Binding Sites of GAPDH --- p.12 / Chapter 1.7 --- Isolation And Purification of GAPDH from Different Organisms --- p.13 / Chapter 1.8 --- Development of New Purification Method of GAPDH Using Immobilized Metal Affinity Chromatography --- p.15 / Chapter 1.9 --- Study of GAPDH from Chironomidae Larvae --- p.16 / Chapter 1.10 --- Aims of Study --- p.18 / Chapter Chapter Two: --- Methods And Materials --- p.19 / Chapter 2.1 --- Isolation of Native Chironomidae GAPDH --- p.19 / Chapter 2.1.1 --- Chemicals And Reagents --- p.19 / Chapter 2.1.2 --- Reagents --- p.19 / Chapter 2.1.3 --- Preparation of Crude Protein Extract from Chironomidae Larvae --- p.24 / Chapter 2.1.4 --- Immobilized Metal Affinity Chromatography --- p.24 / Chapter 2.1.5 --- Large Scale Preparation of Crude Protein Extract --- p.25 / Chapter 2.1.6 --- Ammonium Sulfate Fractionation --- p.25 / Chapter 2.1.7 --- Copper Affinity Column Chromatography --- p.26 / Chapter 2.1.8 --- Dye Affinity Column Chromatography --- p.26 / Chapter 2.2 --- Identification of Chironomidae GAPDH --- p.27 / Chapter 2.2.1 --- Chemicals And Reagents --- p.27 / Chapter 2.2.2 --- Reagents --- p.28 / Chapter 2.2.3 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.32 / Chapter 2.2.4 --- Non-Denaturing Polyacrylamide Gel Electrophoresis --- p.33 / Chapter 2.2.5 --- Protein Extraction from Coosmassie Blue Stained Polyacrylamide Gel --- p.33 / Chapter 2.2.6 --- N-terminal Amino Acid Analysis --- p.33 / Chapter 2.2.7 --- Sequence Analysis --- p.34 / Chapter 2.3 --- Kinetic Analysis of Chironomidae GAPDH --- p.34 / Chapter 2.3.1 --- Chemcials And Reagents --- p.34 / Chapter 2.3.2 --- Reagents --- p.34 / Chapter 2.3.3 --- Determination of Enzyme Concentration And GAPDH Activity --- p.35 / Chapter 2.4 --- Molecular Cloning of Chironomidae GAPDH --- p.36 / Chapter 2.4.1 --- Chemicals And Reagents --- p.36 / Chapter 2.4.2 --- Reagents --- p.37 / Chapter 2.4.3 --- RNA Extraction from Chironomidae Larvae --- p.41 / Chapter 2.4.4 --- DNase I Removal of Genomic DNA Contamination --- p.42 / Chapter 2.4.5 --- RNA Concentration Determination And RNA Agarose Electrophoresis --- p.42 / Chapter 2.4.6 --- First Strand cDNA Synthesis --- p.43 / Chapter 2.4.7 --- pRSet A B C Vectors --- p.43 / Chapter 2.4.8 --- Cloning Primer Design --- p.45 / Chapter 2.4.9 --- Polymerase Chain Reaction --- p.45 / Chapter 2.4.10 --- DNA Agarose Electrophoresis --- p.46 / Chapter 2.4.11 --- Restriction Enzyme Digestion of Insert And Plasmid --- p.46 / Chapter 2.4.12 --- Ligation of Plasmid And Insert DNA --- p.46 / Chapter 2.4.13 --- Preparation of Chemically Competent E. coli --- p.47 / Chapter 2.4.14 --- Transformation of Plasmid by Heat Shock --- p.47 / Chapter 2.4.15 --- Colony PCR --- p.48 / Chapter 2.5 --- Recombinant Protein Expression And Purification --- p.48 / Chapter 2.5.1 --- Chemicals And Reagents --- p.48 / Chapter 2.5.2 --- Reagents --- p.49 / Chapter 2.5.3 --- Protein expression by IPTG --- p.51 / Chapter 2.5.4 --- Protein purification by Nickel Affinity Column Chromatography --- p.52 / Chapter 2.5.5 --- EnterokinaseMax ´ёØ Removal of Polyhistidine Fusion Tag --- p.52 / Chapter 2.5.6 --- Western Blotting of Protein --- p.53 / Chapter Chapter Three: --- Results --- p.54 / Chapter 3.1 --- Two Affinity Chromatography Methods for GAPDH Purification --- p.54 / Chapter 3.2 --- Isolation And Purification of Native Chironomidae GAPDH --- p.54 / Chapter 3.3 --- Identification of Chironomidae GAPDH --- p.62 / Chapter 3.3.1 --- N-terminal amino acid analysis --- p.62 / Chapter 3.3.2 --- Sequence Analysis --- p.62 / Chapter 3.4 --- Molecular Cloning of Chironomidae GAPDH --- p.63 / Chapter 3.5 --- Isolation And Purification of recombinant Chironomidae GAPDH --- p.70 / Chapter 3.6 --- Protein Gel Electrophoresis Analysis of GAPDHs --- p.74 / Chapter 3.7 --- "Effects of Heavy Metals, pH And Temperature on GAPDHs" --- p.76 / Chapter 3.7.1 --- Heavy Metal Effect --- p.76 / Chapter 3.7.2 --- pH Effect --- p.76 / Chapter 3.7.3 --- Temperature --- p.77 / Chapter 3.8 --- Kinetic Analysis of GAPDHs --- p.84 / Chapter Chapter Four: --- Discussion --- p.89 / Chapter 4.1 --- New Method for The Isolation and Purification of Chironomidae GAPDH --- p.89 / Chapter 4.2 --- "Effects of Heavy Metals, pH And Temperature on GAPDHs" --- p.91 / Chapter 4.3 --- Kinetic Analysis of GAPDHs --- p.91 / Chapter 4.4 --- Zinc Activation of Chironomidae GAPDH --- p.92 / Chapter 4.5 --- Future Study --- p.93 / Chapter 4.5.1 --- Sequence Analysis Using Prediction Programmes --- p.94 / Chapter 4.5.2 --- Protein Crystallization --- p.95 / Chapter 4.5.3 --- Site-Directed Mutagenesis --- p.95 / Chapter 4.5.4 --- Biacore Surface Plasmon Resonance --- p.95 / Chapter Chapter Five: --- Conclusion --- p.98 / Chapter Chapter Six: --- References --- p.99

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.