Engineering The Substrate Specificity Of Human Deoxycytidine Kinase Público

Abstract

Human deoxycytidine kinase (dCK) is responsible for the phosphorylation of a number of clinically important nucleoside analog (NA) prodrugs in addition to its natural substrates, 2'-deoxycytidine, 2'-deoxyguanosine, and 2'-deoxyadenosine. To improve the catalytic activity and tailor the substrate specificity of dCK, libraries of mutant enzymes were constructed and tested for thymidine kinase activity. Random and site-saturation mutagenesis were employed to probe for residue positions with an impact on substrate specificity, identifying positions Arg104 and Asp133 in the active site as key residues for substrate specificity. The results illuminate the key contributions of these two amino acids to enzyme function by demonstrating their ability to moderate substrate specificity. Clinically relevant NAs mostly differ from their endogenous counterparts in respect to various modifications at the ribose moiety. To determine the molecular aspects of modified ribose binding, site-saturation mutagenesis was utilized at six amino acid positions in the active site of dCK. All the library members were evaluated by an in vivo screening to find a variant with improved turnover rate for AZT. Kinetic analysis of the selected mutants indicates that the enhanced AZT activity could not be fulfilled with the chosen methodology. Therefore, novel screening methods with positive selection should be developed for further engineering with improved NA phosphorylation. Besides the active site residues, there are other structural elements in the dCK structure that determines the substrate specificity. To test this hypothesis, chimeragenesis was utilized using dCK and the C-terminal region of Dm-dNK to probe the role of phosphoryl donor binding loop in regards to phosphoryl donor and acceptor specificity in dCK. Kinetic analysis of the chimeras indicates that the phosphoryl donor preference was reversed from UTP to ATP as a result of swapped subdomains from Dm-dNK. In order to further investigate the function of donor base-sensing loop, two specific amino acid positions Asp241 and Phe242 were selected for alanine scanning mutagenesis in dCK. The results elucidate the sequence dependency of phosphoryl donor preference and the structure-function relationship in this small phosphoryl donor binding loop region.

Table of Contents

Chapter 1 General Introduction...........................................................................1 1.1 Protein engineering...............................................................................2 1.2 Synthesis of endogenous DNA precursors De novo versus salvage pathway..........................................................3 1.3 Deoxyribonucleoside kinases...............................................................5 1.3.1 Human deoxyribonucleoside kinases............................................6 1.3.2 Multisubstrate insect deoxyribonucleoside kinases......................8 1.3.3 Viral thymidine kinases................................................................9 1.3.4 Bacterial deoxyribonucleoside kinases........................................10 1.4 Type 1 deoxyribonucleoside kinases...................................................10 1.4.1 Basic structural properties of type 1 dNKs..................................11 1.4.2 Substrate specificity and functional versatility link to structural compliance of type 1 dNKs..........................................12 1.5 The role of type 1 dNKs in therapy of diseases....................................15 1.5.1 Nucleoside analogs in chemotherapy and antiviral therapy.........15 1.5.2 Suicide gene/chemotherapy..........................................................18 1.6 Previous engineering studies on dCK ..................................................19 1.7 Aims and scope of the presented dissertation.......................................22 References......................................................................................................24 Chapter 2 Functional investigation of active site mutants responsible for modulating the human deoxycytidine kinase substrate specificity 37

Document Outline Distribution Agreement Acceptance page Abstract Cover Page Abstract Cover Page ACKNOWLEDGEMENTS Table of Contents Final Thesis2

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School	Laney Graduate School
Department	Chemistry
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry
Palabra Clave	Enzyme engineering Anticancer Nucleoside analog Kinase Antiviral therapy
Committee Chair / Thesis Advisor	Lutz, Stefan, Emory University
Committee Members	Liotta, Dennis C, Emory University Conticello, Vincent, Emory University

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