HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 28, 26492-26498, July 13, 2001

This Article Full Text Full Text (PDF) All Versions of this Article: 276/28/26492    most recent M102612200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Marks, D. L. Articles by Pagano, R. E. Search for Related Content PubMed PubMed Citation Articles by Marks, D. L. Articles by Pagano, R. E. Social Bookmarking                      What's this?

Identification of Active Site Residues in Glucosylceramide Synthase
A NUCLEOTIDE-BINDING/CATALYTIC MOTIF CONSERVED WITH PROCESSIVE -GLYCOSYLTRANSFERASES^*

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

From the Mayo Clinic and Foundation, Thoracic Diseases Research Unit, Department of Biochemistry and Molecular Biology, Rochester, Minnesota 55905

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 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 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 Cys-207 was the primary residue involved in GCS N-ethylmaleimide sensitivity. Next, we showed by multiple alignment that the region of GCS flanking His-193 and Cys-207 (amino acids 89-278) contains a D1,D2,D3,(Q/R)XXRW motif found in the putative active site of processive -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 -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 -glycosyltransferases possess a conserved substrate-binding/catalytic domain.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				A. E. Ciocchini, M. S. Roset, G. Briones, N. I. de Iannino, and R. A. Ugalde Identification of active site residues of the inverting glycosyltransferase Cgs required for the synthesis of cyclic {beta}-1,2-glucan, a Brucella abortus virulence factor Glycobiology, July 1, 2006; 16(7): 679 - 691. [Abstract] [Full Text] [PDF]

				G. Holzl, M. Leipelt, C. Ott, U. Zahringer, B. Lindner, D. Warnecke, and E. Heinz Processive lipid galactosyl/glucosyltransferases from Agrobacterium tumefaciens and Mesorhizobium loti display multiple specificities Glycobiology, September 1, 2005; 15(9): 874 - 886. [Abstract] [Full Text] [PDF]

				Y.-Y. Liu, T. Y. Han, J. Y. Yu, A. Bitterman, A. Le, A. E. Giuliano, and M. C. Cabot Oligonucleotides blocking glucosylceramide synthase expression selectively reverse drug resistance in cancer cells J. Lipid Res., May 1, 2004; 45(5): 933 - 940. [Abstract] [Full Text] [PDF]

				H. van der Wel, S. Z. Fisher, and C. M. West A Bifunctional Diglycosyltransferase Forms the Fucalpha 1,2Galbeta 1,3-Disaccharide on Skp1 in the Cytoplasm of Dictyostelium J. Biol. Chem., November 22, 2002; 277(48): 46527 - 46534. [Abstract] [Full Text] [PDF]

				J. Stolz and S. Munro The Components of the Saccharomyces cerevisiae Mannosyltransferase Complex M-Pol I Have Distinct Functions in Mannan Synthesis J. Biol. Chem., November 15, 2002; 277(47): 44801 - 44808. [Abstract] [Full Text] [PDF]

				P. E. Pummill and P. L. DeAngelis Evaluation of Critical Structural Elements of UDP-Sugar Substrates and Certain Cysteine Residues of a Vertebrate Hyaluronan Synthase J. Biol. Chem., June 7, 2002; 277(24): 21610 - 21616. [Abstract] [Full Text] [PDF]

				M. Leipelt, D. Warnecke, U. Zahringer, C. Ott, F. Muller, B. Hube, and E. Heinz Glucosylceramide Synthases, a Gene Family Responsible for the Biosynthesis of Glucosphingolipids in Animals, Plants, and Fungi J. Biol. Chem., August 31, 2001; 276(36): 33621 - 33629. [Abstract] [Full Text] [PDF]