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

Glucosylceramide synthase (GCS) transfers glucose from UDP-Glc to ceramide, catalyzing the first glycosylation step in the formation of higher order g lycosphingolipids, The amino acid sequence of GCS was reported to be dissim ilar from other proteins, with no identifiable functional domains. We previ ously identified His-193 of rat GCS as an important residue in UDP-Glc and GCS inhibitor binding; however, little else is known about the GCS active s ite. Here, we identify key residues of the GCS active site by performing bi ochemical and site-directed mutagenesis studies of rat GCS expressed in bac teria. First, we found that Cys-207 was the primary residue involved in GCS N-ethylmaleimide sensitivity. Next, we showed by multiple alignment that t he region of GCS flanking His-193 and Cys-SO? (amino acids 89-278) contains a D1,D2,D3,(Q/R)XXRW motif found in the putative active site of processive beta -glycosyltransferases (e,g, cellulose, chitin, and hyaluronan synthes es). Site-directed mutagenesis studies demonstrated that most of the highly conserved residues were essential for GCS activity. We also note that GCS and processive beta -glycosyltransferases are topologically similar, posses sing cytosolic active sites, with putative transmembrane domains immediatel y N-terminal to the conserved domain. These results provide the first exten sive information on the GCS active site and show that GCS and processive be ta -glycosyltransferases possess a conserved substrate-binding/catalytic do main.