Structural effects of naturally occurring human blood group B galactosyltransferase mutations adjacent to the DXD motif.
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 (211)DVD(213) motif that coordinates to a Mn(2+) ion shown to be critical in donor binding and catalysis. Three GTB mutants, M214V, M214T, and M214R, with alterations adjacent to the (211)DVD(213) motif have been identified in blood banking laboratories. From serological phenotyping, individuals with the M214R mutation show the B(el) 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 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 Mn(2+). 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.
Persson
Mattias
M
Carlsberg Laboratory, Gamle Carlsberg Vej 10, 2500 Valby, Copenhagen, Denmark.
Letts
James A
JA
Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada.
Hosseini-Maaf
Bahram
B
Division of Hematology and Transfusion Medicine, Department of Laboratory Medicine, Lund University and The Blood Centre, Lund University Hospital, SE-22185 Lund, Sweden.
Borisova
Svetlana N
SN
Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada.
Palcic
Monica M
MM
Carlsberg Laboratory, Gamle Carlsberg Vej 10, 2500 Valby, Copenhagen, Denmark. Electronic address: monica@crc.dk.
Evans
Stephen V
SV
Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada.
Olsson
Martin L
ML
Division of Hematology and Transfusion Medicine, Department of Laboratory Medicine, Lund University and The Blood Centre, Lund University Hospital, SE-22185 Lund, Sweden. Electronic address: Martin_L.Olsson@med.lu.se.
D000017:ABO Blood-Group System / Q000737:chemistry* / Q000235:genetics*; D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D002384:Catalysis; D018360:Crystallography, X-Ray; D005700:Galactosyltransferases / Q000097:blood / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D008715:Methionine / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics
D000017:ABO Blood-Group System; D008715:Methionine; D005700:Galactosyltransferases; C023155:blood-group-substance alpha-D-galactosyltransferase