The DXD motif is required for GM2 synthase activity but is not critical for nucleotide binding.
We tested the importance of the aspartate-any residue-aspartate (DXD) motif for the enzymatic activity and nucleotide binding capacity of the Golgi glycosyltransferase GM2 synthase. We prepared point mutations of the motif, which is found in the sequence 352-VLWVDDDFV, and analyzed cells that stably expressed the mutated proteins. Whereas the folding of the mutated proteins was not seriously disrupted as judged by assembly into homodimers, Golgi localization, and secretion of a soluble form of the enzyme, exchange of the highly conserved aspartic acid residues at position 356 or 358 with alanine or asparagine reduced enzyme activity to background levels. In contrast, the D356E and D357N mutations retained weak activity, while the activity of V352A and W354A mutants was 167% and 24% that of wild-type enzyme, respectively. Despite the major effect of the DXD motif on enzymatic activity, nucleotide binding was not altered in the triple mutant D356N/D357N/D358N as revealed by binding to UDP-beads and labeling with the photoaffinity reagent, P(3)-(4-azidoanilido)uridine 5'-triphosphate (AAUTP). In summary, rather than being critical for nucleotide binding, this motif may function during catalysis in GM2 synthase, as has been proposed elsewhere for the SpsA glycosyltransferase based on its crystal structure.
Li
J
J
Department of Molecular, Cellular, and Craniofacial Biology, School of Medicine, University of Louisville, 501 S. Preston St., Louisville, KY 40292, USA.
Rancour
D M
DM
Allende
M L
ML
Worth
C A
CA
Darling
D S
DS
Gilbert
J B
JB
Menon
A K
AK
Young
W W
WW
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005434:Flow Cytometry; D009154:Mutation; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D019920:Photoaffinity Labels; D011485:Protein Binding
D009711:Nucleotides; D019920:Photoaffinity Labels; D017350:N-Acetylgalactosaminyltransferases; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase