Alpha-Galactosidase (alpha-GAL) purified from green coffee bean cleaves the terminal galactose residue from the surface of group B erythrocytes, thereby converting these cells serologically to group O cells. A sufficient supply of """"""""universal"""""""" blood, as group O blood is known, will have a significant impact on the blood transfusion medicine. However, little is known about how alpha-GAL functions at a molecular level and critical amino acid residues affect its catalytic mechanism. Here, we propose the study of structure-function relationship of alpha-GAL with the following specific objectives. (1) The first objective is to identify critical residues involved in the catalytic mechanism for the understanding of the structure-function relationship of alpha-GAL. The conserved residues based on sequence homology will be chosen for site-directed mutagenesis. Mutated alpha- GALs, expressed in P. pastoris, will be characterized in order to identify critical residues in the active site and to determine their roles in the enzymatic mechanism. Furthermore, the understanding of the structure- function relationship of the enzyme will be facilitated as a result of a collaborative study of X-ray crystallography of coffee bean alpha-GAL. The data derived from these studies will provide a """"""""rational"""""""" guideline for substitutions of amino acid residues at or near the active site in order to alter the enzymatic properties desired for the blood-type conversion reaction. (2) The second objective is to apply random mutagenesis for functional alteration of the alpha-GAL molecule in order to improve its properties for B yields O blood group conversion. An efficient two-step assay system will be established, which can be used for screening a large number of transformed colonies and directly measuring the enzymatic activity towards the terminal galactose residues on the red cell surface under variety of conditions. Randomly mutated alpha-GAL will be expressed in P. pastoris and then screened for mutants with the desired properties, including significant activity at physiological pH, high turnover number and high thermostability. Furthermore, amino acid residues in the enzyme mutants which are responsible for these functional alterations will be localized and characterized.
