TY - JOUR
T1 - Structural effects of naturally occurring human blood group B galactosyltransferase mutations adjacent to the DXD motif
AU - Persson, Mattias
AU - Letts, James A.
AU - Hosseini Maaf, Bahram
AU - Borisova, Svetlana N.
AU - Palcic, Monica M.
AU - Evans, Stephen V.
AU - Olsson, Martin L
PY - 2007
Y1 - 2007
N2 - 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 (Fuc alpha(1-2)Gal beta-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 (DVD213)-D-211 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 (211)DVD213 motif have been identified in blood banking laboratories. From serological phenotyping, individuals with the M214R mutation show the B,1 variant expressing very low levels of B antigens, whereas those with M214T and M214V mutations give rise to A(weak)B phenotypes. Kinetic analysis of recombinant mutant GTB enzymes revealed that M214R has a 1200-fold decrease in k(cat) compared with wild type GTB. The crystal structure of M214R showed that DVD motif coordination to Mn2+ 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.
AB - 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 (Fuc alpha(1-2)Gal beta-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 (DVD213)-D-211 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 (211)DVD213 motif have been identified in blood banking laboratories. From serological phenotyping, individuals with the M214R mutation show the B,1 variant expressing very low levels of B antigens, whereas those with M214T and M214V mutations give rise to A(weak)B phenotypes. Kinetic analysis of recombinant mutant GTB enzymes revealed that M214R has a 1200-fold decrease in k(cat) compared with wild type GTB. The crystal structure of M214R showed that DVD motif coordination to Mn2+ 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.
U2 - 10.1074/jbc.M610998200
DO - 10.1074/jbc.M610998200
M3 - Article
C2 - 17259183
SN - 1083-351X
VL - 282
SP - 9564
EP - 9570
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 13
ER -