The role of the carboxyl (C)-terminal region of coffee bean alpha-galactosidase (alpha-GAL) has been studied by expressing C-terminal deletion mutants in the methylotrophic yeast strain Pichia pastoris. A previous study of human alpha-galactosidase determined that enzyme activity increased when up to 10 amino acid residues were deleted. Deleting 11 residues reduced activity, and deleting 12 residues abolished activity. In our studies, alpha-GAL activity is reduced when one or two amino acids are deleted, as is enzyme secretion directed by P. pastoris signal sequences. The pH profile is similar to that of the wild-type enzyme. Deleting 3 or more residues from the C-terminal end results in a complete loss of both enzyme secretion and activity. The C-terminus of alpha-GAL seems to play an important role in overall enzyme conformation and may directly affect the proper conformation of the active site.
Zhu A, Leng L, Monahan C, Zhang Z, Hurst R, et al.
Archives of biochemistry and biophysics •
Alpha-Galactosidase (alpha-GAL) purified from green coffee bean cleaves the terminal galactose residues from the surface of group B erythrocytes, thereby converting these cells serologically to group O cells. Such enzymatically converted red cells have been transfused into group A and O recipients as part of the first phase of FDA-approved clinical trials. Recently we expressed the recombinant alpha-GAL (r)alpha-GAL) in large quantities in a methylotrophic yeast strain Pichia pastoris and purified the protein to apparent homogeneity by chromatography on a macro prep S50 column. Purified (r)alpha-GAL, migrating as a single band of 41 kDa on a SDS-PAGE, appears to be identical to its native counterpart in specific activity (32 U/mg) and kinetic parameters (K(m) =0.363 mM and V(max) = 46.9 U/mg). Both enzymes demonstrate the same pH profile in the pH range from 2 to 9, with an optimal pH at 6.4 when tested with the substrate p-nitrophenol-alpha-D-galactopyranoside. Furthermore, as with its native counterpart, (r)alpha-GAL specifically cleaves alpha-linked terminal galactose residues from group B red cells without affecting other major antigens on the red cell surface. In addition, we developed a method for using RT-PCR to detect possible DNA contamination in the purified protein preparation, which is one of the concerns for in vivo studies. Thus, with a simple procedure for over-expression and purification of (r)alpha-GAL from P. pastoris culture, one can readily obtain the enzyme needed for large-scale sero-conversion of red cells.
The cDNA for coffee bean alpha-galactosidase (alpha-Gal) has been cloned and expressed in a baculovirus expression system. An early study of coconut alpha-Gal by chemical modification suggested that one tyrosine residue is at or near the active site. In order to identify such a critical residue, we replaced two tyrosine residues (positions 108 and 158) with phenylalanine by site-directed mutagenesis. The mutated DNA strands, as well as the wild-type ones, were subcloned into pVL vector and transformed into Sf9 insect cells for intracellular expression. The replacement of Tyr-158 with phenylalanine resulted in a mutant alpha-Gal (Y158F) which retained approx. 88% of the activity of wild-type enzyme. However, the substitution of Tyr-108 by phenylalanine (Y108F) almost abolished the enzymatic activity (1.8% of wild-type activity). The Vmax/Km value for the mutant Y108F was 0.027, which was over a 1000-fold lower than that of wild-type alpha-Gal. Our data suggest that Tyr-108 is critical for the enzymatic activity of alpha-Gal.
Purified coffee bean alpha-galactosidase (alpha Gal) has been used for removing terminal alpha-galactose residues from the glyco-conjugates at the red cell surface, in studies of blood group conversion. Here, we report the isolation and sequence of the full-length clone for coffee bean alpha Gal by using the polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) techniques. The cDNA clone (1.4 kb) contains a single open reading frame which encodes a protein of 378 amino acids (aa). Its authenticity is confirmed by perfect alignment of aa sequences obtained from purified coffee bean alpha Gal, and by immune reaction with the antibody raised against the enzyme. Furthermore, the protein produced in insect cells shows enzymatic activity towards a synthetic alpha Gal substrate, p-nitro-phenyl-alpha-galactopyranoside.
