Structure-function Analysis of Human alpha1,3-fucosyltransferase. Amino Acids Involved in Acceptor Substrate Specificity

J Biol Chem. 1996 Apr 12;271(15):8818-23. doi: 10.1074/jbc.271.15.8818.

Abstract

A series of molecular biology experiments were carried out to identify the catalytic domain of two human alpha1,3/4-fucosyltransferases (fucosyltransferases (FucTs) III and V), and to identify amino acids that function in acceptor substrate binding. Sixty-one and 75 amino acids could be eliminated from the N terminus of FucTs III and V, respectively, without a significant loss of enzyme activity. In contrast, the truncation of one or more amino acids from the C terminus of FucT V resulted in a dramatic or total loss of enzyme activity. Results from the truncation experiments demonstrate that FucT III62-361 (containing amino acids 62-361) and FucT V76-374 (containing amino acids 76-374) are active, whereas shorter forms of the enzymes were inactive. The shortest, active forms of the enzymes are more than 93% identical at the predicted amino acid level, but have distinct acceptor substrate specificities. Thus, FucT III is an alpha1,4-fucosyltransferase, whereas FucT V is an alpha1,3-fucosyltransferase with disaccharide substrates. All but one of the amino acid sequence differences between the two proteins occur near their N terminus. Results obtained from domain swapping experiments demonstrated that the single amino acid sequence difference near the C terminus of these enzymes did not alter the enzyme's substrate specificity. However, swapping a region near the N terminus of the truncated form of FucT III into an homologous region in FucT V produced a protein with both alpha1,3- and alpha1,4-fucosyltransferase activity. This region contains 8 of the amino acid sequence differences that occur between the two proteins.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • DNA Primers / chemistry
  • Fucosyltransferases / chemistry
  • Fucosyltransferases / metabolism*
  • Humans
  • Molecular Sequence Data
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Sequence Deletion
  • Structure-Activity Relationship
  • Substrate Specificity

Substances

  • DNA Primers
  • Recombinant Fusion Proteins
  • Fucosyltransferases
  • galactoside 3-fucosyltransferase