Nucleoside hydrolases are a family of structurally related metalloproteins, which were initially identified and characterized in parasitic protozoan where they have a well-established metabolic role in the nucleoside salvage pathways. Due to their vital importance in these pathogens, parasitic NHs have been extensively studied by X-ray crystallography and kinetic methods as potential targets for chemotherapeutic treatment. Nucleoside hydrolase activity has also been identified but poorly characterized in a wide range of organisms differently positioned in the evolution tree of life like bacteria, yeast, insects, and mesozoa, where however their precise functional role is still debated. More detailed studies, especially on their substrate specificity, may reveal the true physiological function of the non-protozoan NHs. Recently the bacterial Y eiK NH from E. coli has been cloned and expressed as a recombinant protein. The analysis of its substrate specificity pattern indicates the existence of a class of hydrolases that differs from the already characterized purine specific and "nonspecific" NHs in their strict preference for the pyrimidine nucleosides. Moreover from the crystal structure has been shown that active site common features and some differences with the other NHs could explain the Y eiK absolute specificity for pyrimidines. The initial aim of this work has been the structural and functional characterization of other two pyrimidine-specific NH enzymes in order to extend the knowledge on this class of NHs and clarify their catalytic mechanism. The approach has been to carry out in parallel the biochemical and kinetic studies with the crystallization and X-ray structure determination of both the proteins. The enzymatic properties of YbeK and URHl have confirmed their specificity towards pyrimidine nucleosides, and have moreover highlighted for the yeast enzyme a high catalytic efficiency in converting nucleoside analogues that are prodrugs used in the treatment of cancer. The advantage of the nucleoside analogues over the base analogues is that they are more bioavailable and that usually cancer cells develop a less pronounced resistance to their action. These drugs require an enzymatic conversion to the active form by cellular enzymes, which result to be a common limiting step and an efficacy loss reason. The substrate specificity and catalytic efficiency of URHl, together with its biochemical features, have highlighted its possible application as a suicide gene in cancer gene therapy. With this goal in mind, a bifunctional chimeric protein has been designed to improve the effect and limit the toxicity of the nucleoside analogues. The chimeric enzyme couples the pyrimidine hydrolase activity of URHl with the phosphoribosyltransferase activity of the E. coli UPRTase enzyme in order to directly and efficiently convert the 5-fluouridine prodrug in its 5-fluorouracil monophosphate active form. The high resolution three dimensional structure of YbeK that is presented in this thesis leads to a better understanding of the structure-function relationships for the pyrimidinespecific enzymes, which will be useful to improve through rationally designed mutagenesis the enzymatic catalytic efficiency of NHs. Together with the promising results obtained with the chimeric bifunctional URHl-UPRTase protein, this work paves the way to a possible clinical application of NHs in cancer gene therapy. The details of the experimental methods employed and the results obtained to achieve the initial aim are here described and discussed. This work is part of a collaboration between the Biocrystallography Unit of the DIBIT, S. Raffaele Scientific Institute in Milan, where I performed the experimental work, and the Laboratorium voor Ultrastructure and Molecular Biologie directed by Dr. Jan Steyaert at the Vrije Universiteit in Brussels.
|Titolo:||Structural and functional characterization of two pyrimidine nucleoside hydrolases, novel enzymes for cancer suicide gene therapy|
|Relatore/i esterni:||Degano, Massimo|
|Data di pubblicazione:||29-giu-2004|
|Appare nelle tipologie:||8.1 PhD thesis|