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dc.contributor.authorGopaul, Deshmukh Neerunjun
dc.date.accessioned2018-07-12T18:50:15Z
dc.date.available2018-07-12T18:50:15Z
dc.date.issued1997
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 58-01, Section: B, page: 1820.;Advisors: James C. Sacchettini; Vern L. Schramm.
dc.identifier.urihttps://yulib002.mc.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:9717816
dc.identifier.urihttps://hdl.handle.net/20.500.12202/3706
dc.description.abstractThe purine salvage pathway is an important target for anti-protozoal drug design due to the absence of de novo synthetic routes in parasitic protozoans. IU-nucleoside hydrolase (IU-NH) catalyzes the hydrolysis of nucleosides through an oxy-carbenium transition state. The attribution of functionality in an enzyme's active-site can be achieved through the determination its three-dimensional structure by x-ray crystallography. The gene for IU-NH has been cloned from genomic DNA by reverse-transcription/PCR of polyA m-RNA from C. fasciculata, using an approach based on the mini-exon sequence and oligo-nucleotides for internal peptides. The primary structure consist of 315 amino acids and agrees with the predicted {dollar}\rm M\sb{lcub}r{rcub} = 34,194{dollar} Da. There is significant homology (an average of 37% in identity) with open reading frames from a wide variety of species, including yeast and a homolog from the human pathogen, Leishmania major. The recombinant enzyme produced by over-expression in E. coli cells, and native enzyme, have both been successfully purified to homogeneity and crystallized. The three-dimensional structure of the enzyme is reminiscent of a nucleotide binding protein, with a core region similar to a Rossmann fold. Conserved clusters of aspartic acid residues are involved in transition state stabilization and binding of a {dollar}\rm Ca\sp{lcub}2+{rcub}{dollar}-metal ion. Site-directed mutagenesis has identified His241 as the proton donor, Asp10 as a metal-ion ligand and the possible active-site base, and Glu166 as a residue crucial for catalysis. The structural information from C. fasciculata will identify targets in other pathogenic protozoa, and will be essential in improving the affinity of inhibitors for IU-nucleoside hydrolase.
dc.publisherProQuest Dissertations & Theses
dc.subjectBiochemistry.
dc.subjectMolecular biology.
dc.subjectPharmacy sciences.
dc.titleThe inosine-uridine nucleoside hydrolase from Crithidia fasciculata. Genetic, crystallization, and functional studies
dc.typeDissertation


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