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M102612200v1
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Papers In Press, published online ahead of print May 3, 2001
J. Biol. Chem, 10.1074/jbc.M102612200
Submitted on March 22, 2001
Revised on May 1, 2001
Accepted on May 3, 2001

Identification of active site residues in glucosylceramide synthase: A nucleotide-binding/catalytic motif conserved with processive ß-glycosyltransferases

David L. Marks, Michel Dominguez, Kangjian Wu, and Richard E. Pagano

Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, MN 55905

Corresponding Author: pagano.richard{at}mayo.edu

Glucosylceramide synthase (GCS) transfers glucose from UDP-Glc to ceramide, catalyzing the first glycosylation step in the formation of higher order glycosphingolipids. The amino acid sequence of GCS was reported to be dissimilar from other proteins, with no identifiable functional domains. We previously identified H193 of rat GCS as an important residue in UDP-Glc and GCS inhibitor binding; however, little else is known about the GCS active site. Here, we identify key residues of the GCS active site by performing biochemical and site-directed mutagenesis studies of rat GCS expressed in bacteria. First, we found that C207 was the primary residue involved in GCS NEM sensitivity. Next, we showed by multiple alignment that the region of GCS flanking H193 and C207 (amino acids 89-278) contains a D1,D2,D3, Q/RXXRW motif found in the putative active site of processive ß-glycosyltransferases (e.g., cellulose, chitin and hyaluronan synthases). Site-directed mutagenesis studies demonstrated that most of the highly conserved residues were essential for GCS activity. We also note that GCS and processive ß-glycosyltransferases are topologically similar, possessing cytosolic active sites, with putative transmembrane domains immediately N-terminal to the conserved domain. These results provide the first extensive information on the GCS active site, and show that GCS and processive ß-glycosyltransferases possess a conserved substrate binding/catalytic domain.


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