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Towards universal blood : mechanistic studies on blood group cleaving glycosidases

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Title: Towards universal blood : mechanistic studies on blood group cleaving glycosidases
Author: Shaikh, Fathima Aidha
Degree Doctor of Philosophy - PhD
Program Chemistry
Copyright Date: 2012
Publicly Available in cIRcle 2012-08-23
Abstract: The ABO blood groups are vitally important in blood transfusion and organ transplantation. Transfusion with an incorrect blood type results in destruction of the incompatible blood cells, which can result in death. In my thesis, the catalytic mechanisms of three enzymes, two of which can directly be used on red blood cells (RBCs), were investigated in detail as follows. YesZ, a family GH 42 β-galactosidase (retaining), was used as a model system for the identification of catalytic residues. The mechanism-based inhibitor, 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-D-galactopyranoside was synthesized and used to inactivate YesZ via trapping of a reaction intermediate. Subsequent proteolytic digestion and comparative MS analysis identified the labeled peptide which, combined with, sequence alignments identified the catalytic nucleophile, a glutamate in the sequence ETSPSYAASL. Use of the acid/base mutant for trapping experiments provided support for its role thereby providing experimental verification of the identities of the catalytic residues in Family GH42. EABase, a family GH98 endo-β-galactosidase, cleaves blood group A and B trisaccharides from glycoconjugates and RBCs. The mechanism of Family 98 glycosidases was unknown but inferred to be retaining. The DNP-β-A-trisaccharide substrate was synthesized by in vivo enzymatic and subsequent chemical methods and direct 1H NMR analysis of its hydrolysis by EABase revealed that EABase is an inverting glycosidase. Both activated and nonactivated substrates were used to kinetically characterize EABase and its mutants, revealing that D453 and/or E506 act as the base catalyst and that E354 is the acid catalyst. EABase was used, in collaboration with Dr. Kizhakkedathu’s lab, to generate “universal blood cells” from type-B blood. Several α-L-fucosidases from family GH29 (retaining), which cleave α(1,2)-fucose from glycoconjugates were kinetically characterized in the hope of identifying the acid/base residue which is not conserved by sequence. A combination of modeling, sequence comparisons and phylogenetic tree analysis was used to identify candidate acid/base residues and further subgroup GH29 fucosidases based on these comparisons. The identity of the acid/base residue in four fucosidases is supported by kinetic characterization of a series of mutants of candidate residues and can now be predicted for all Family GH29 fucosidases.
URI: http://hdl.handle.net/2429/43027
Scholarly Level: Graduate

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