Structural effects of naturally occurring human blood group B galactosyltransferase mutations adjacent to the DXD motif

Abstract

Human blood group A and B antigens are produced by two closely related glycosyltransferase enzymes. An N-acetylgalactosaminyltransferase (GTA) utilizes UDP-GalNAc to extend H antigen acceptors (Fucα(1-2)Galβ-OR) producing A antigens, whereas a galactosyltransferase (GTB) utilizes UDP-Gal as a donor to extend H structures producing B antigens. GTA and GTB have a characteristic 211DVD213 motif that coordinates to a Mn2+ ion shown to be critical in donor binding and catalysis. Three GTB mutants, M214V, M214T, and M214R, with alterations adjacent to the 211DVD213 motif have been identified in blood banking laboratories. From serological phenotyping, individuals with the M214R mutation show the Bel variant expressing very low levels of B antigens, whereas those with M214T and M214V mutations give rise to AweakB phenotypes. Kinetic analysis of recombinant mutant GTB enzymes revealed that M214R has a 1200-fold decrease in kcat compared with wild type GTB. The crystal structure of M214R showed that DVD motif coordination to Mn 2+ was disrupted by Arg-214 causing displacement of the metal by a water molecule. Kinetic characterizations of the M214T and M214V mutants revealed they both had GTA and GTB activity consistent with the serology. The crystal structure of the M214T mutant showed no change in DVD coordination to Mn2+. Instead a critical residue, Met-266, which is responsible for determining donor specificity, had adopted alternate conformations. The conformation with the highest occupancy opens up the active site to accommodate the larger A-specific donor, UDP-GalNAc, accounting for the dual specificity. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.