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Title: Structural studies of coenzyme-dependent enzymes
Author: Ruane, Karen Mary
ISNI:       0000 0004 2749 6181
Awarding Body: University of York
Current Institution: University of York
Date of Award: 2009
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Enzymes, nature's catalysts, catalyse a diverse array of reactions. In many cases, enzymes only require the 20 amino acids from which they are constructed to carry out a reaction. However there are many examples where an enzyme which carries out a certain class of reaction, such as acyl or hydride transfer, requires an additional chemical group to perform the reaction. These additional groups are called coenzymes. The work described in this thesis primarily covers the structural characterisation of a set of, somewhat disparate, enzymes all of which require coenzymes to carry out their chemistries. Following an introduction to cofactors and their likely evolution from an "RNA world" in Chapter 1, two particular coenzyme-dependent enzymes will be discussed. Chapter 2 covers the structure solution of Pg ID, an acetyltransferase from Campylobacter jejuni in complex with the Coenzyme A portion of the coenzyme acetyl-CoA at 1.8 A resolution. This enzyme catalyses the transfer of an acetyl group onto the substate sugar to produce bacillosamine, an unusual sugar which forms the base from which the N-linked glycan in C. jejuni is built. Chapters 3-5 focus on formate dehydrogenases (FDHs) and in particular their cofactor specificity for NAD+ vs. NADP+. FDHs can be used industrially to recycle NAD+, their cofactor of choice. However in an industrial context, many more industrial biotransformations use NADP+ as cofactor rather than NAD+. Chapter 3 gives an overview of cofactor regeneration and covers the analysis of the "first" NADP+ specific FDH and its 3-D structure at 1.5 A resolution. Chapter 4 focuses on how NADP+ is bound within the active site and probes how the specificity occurs. This is achieved through complex structures of both native BspFDH and a mutant which was designed to reverse the coenzyme specificity of the enzyme to NAD+. Chapter 5 describes the conversion of a formally NAD+ specific FDH into one that equally well uses NADP+ as cofactor. We believe that these enzymes have potential for industrial cofactor regeneration. Additionally, a single wavelength anomalous dispersion structure solution, that of a family GT4 glycosyltransferase, is described in an appendix. This was a very challenging project with -3.1 A data and twelve molecules in the asymmetric unit. The results and the relationship of this enzyme to known glycosyltransferases is discussed. There is also a brief description of the methods used throughout this thesis in a further appendix chapter.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available