Glycobiology Advance Access published online on July 8, 2003
Glycobiology, doi:10.1093/glycob/cwg093
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© 2003 Oxford University Press
ORIGINAL ARTICLES
1 Department of Biochemistry, La Trobe University, Melbourne, Victoria 3086, Australia The crdS gene of Agrobacterium sp. strain ATCC31749 encodes the curdlan synthase protein based on the homology of the derived CrdS protein sequence with those of
Revised on June 5, 2003
Accepted on June 10, 2003
Topological characterization of an inner membrane (1
3)-
-D-glucan (curdlan) synthase from Agrobacterium sp. strain ATCC31749
2 CSIRO Division of Health Sciences & Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia
3 Department of Microbiology, La Trobe University, Melbourne, Victoria 3086, Australia
-glycosyl transferases with repetitive action patterns (Stasinopoulos et al., 1999 Glycobiology, 9, 31-41). Here we show that chemical (NTG)-mutagenesis of crdS abolishes curdlan production and that the induced mutations can be complemented by a cloned crdS amplicon, thus providing genetic confirmation that crdS is essential for curdlan production. When expressed in the native Agrobacterium or in Escherichia coli, the largely hydrophobic CrdS protein exhibited a Mr of 
60 kDa compared with the predicted mass of 73,121 Da, and was located in the inner membrane of both bacteria. By analyzing reciprocal fusions between crdS and the reporter genes, lacZ and phoA, and assessing the sensitivity of CrdS in spheroplasts to proteinase K, CrdS was shown to be an integral membrane protein with seven transmembrane helices and an Nout-Cin disposition. A central large and relatively hydrophilic, cytoplasmic region carries the substrate-binding and catalytic D,D,D35QxxRW motif. The amino acid sequence of this domain of CrdS was threaded onto the three-dimensional structure of the comparable domain of the SpsA protein, a member of the family GT-2 glycosyl transferases, and enabled the identification of corresponding amino acids involved in binding UDP in CrdS. Analysis of Agrobacterium membrane preparations using Blue Native-PAGE provided preliminary evidence that CrdS occurs in multimeric protein complexes of 420 kDa and 500 kDa.
Agrobacterium ATCC31749, curdlan, (1
3)--glucan, (13)--glucan synthase, membrane topology, modelling, multimeric complex, proteinase K sensitivity, reporter gene fusions
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