C-terminal truncation of alpha 1,6-fucosyltransferase from Rhizobium sp. does not annul the transferase activity of the enzyme.
Recently we have over-expressed the enzyme alpha 1,6-fucosyltransferase from Rhizobium sp. in Escherichia coli. In this heterologous system the enzyme was mainly expressed as inclusion bodies and the one that was expressed soluble showed a short-lasting activity in solution due to precipitation of the protein. A structural analysis of the sequence using the TMpred program predicted a highly hydrophobic region of 19 aa close to the C-terminal of the protein. In order to investigate the influence of this region on the formation of inclusion bodies and the precipitation from solution, we cloned a truncated version of the protein where a C-terminal fragment of 65 aa, including the predicted transmembrane-like region, was removed. The resulting protein was expressed in a soluble form without formation of inclusion bodies. The truncated protein catalyzed the transfer of a fucopyranosyl moiety from GDP-beta-L-Fucose to chitobiose. Comparison of the acceptor specificity between the truncated alpha 1,6-fucosyltransferase and the wild-type enzyme, showed a similar behavior for both enzymes. Our results indicate that the active center is not located in the C-terminal extreme of the protein in contrast to the case of the mammalian glycosyltransferases. Also, these results indicate that the alpha-6-motif III is not directly involved in the catalytic activity of the enzyme.
Bastida
Agatha
A
Departamento de Química Orgánica Biológica, Instituto de Química Orgánica General, CSIC, Madrid 28006, Spain.
Fernández-Mayoralas
Alfonso
A
García-Junceda
Eduardo
E
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004187:Disaccharides / Q000378:metabolism; D005647:Fucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006154:Guanosine Diphosphate Fucose / Q000378:metabolism; D002479:Inclusion Bodies / Q000201:enzymology; D008969:Molecular Sequence Data; D012231:Rhizobium / Q000201:enzymology*; D017384:Sequence Deletion; D012995:Solubility; D013329:Structure-Activity Relationship
D004187:Disaccharides; D006154:Guanosine Diphosphate Fucose; C032438:chitobiose; D005647:Fucosyltransferases; C020559:Glycoprotein 6-alpha-L-fucosyltransferase