C-terminal truncation of alpha 1,6-fucosyltransferase from Rhizobium sp. does not annul the transferase activity of the enzyme

Bioorg Med Chem. 2002 Mar;10(3):737-42. doi: 10.1016/s0968-0896(01)00327-3.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Catalytic Domain
  • Cloning, Molecular
  • Disaccharides / metabolism
  • Fucosyltransferases / chemistry*
  • Fucosyltransferases / genetics
  • Fucosyltransferases / metabolism*
  • Guanosine Diphosphate Fucose / metabolism
  • Inclusion Bodies / enzymology
  • Molecular Sequence Data
  • Rhizobium / enzymology*
  • Sequence Deletion
  • Solubility
  • Structure-Activity Relationship

Substances

  • Disaccharides
  • Guanosine Diphosphate Fucose
  • chitobiose
  • Fucosyltransferases
  • Glycoprotein 6-alpha-L-fucosyltransferase