Single amino acid mutations of Medicago glycosyltransferase UGT85H2 enhance activity and impart reversibility

FEBS Lett. 2009 Jun 18;583(12):2131-5. doi: 10.1016/j.febslet.2009.05.046. Epub 2009 Jun 3.

Abstract

The glycosyltransferase UGT85H2 from Medicago truncatula catalyzes glucosylation of the (iso)flavonoids kaempferol and biochanin A. Structure-based mutagenesis of UGT85H2 was carried out to explore the roles of amino acids involved in substrate binding. Substitution of Ile305 by threonine increased catalytic efficiency 37- or 19-fold with kaempferol or biochanin A as acceptor, respectively. A point mutation V200E also dramatically improved the turnover rate and catalytic efficiency by 15-fold for kaempferol and 54-fold for biochanin A. More interestingly, this single mutation (V200E) conferred reversibility in the glycosyltransfer reaction, indicating that Glu200 is a key determinant for the deglycosylation function.

Publication types

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

MeSH terms

  • Amino Acid Substitution
  • Catalytic Domain / genetics
  • Genistein / metabolism
  • Glycosyltransferases / chemistry
  • Glycosyltransferases / genetics*
  • Glycosyltransferases / metabolism*
  • Kaempferols / metabolism
  • Kinetics
  • Medicago truncatula / enzymology*
  • Medicago truncatula / genetics*
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity

Substances

  • Kaempferols
  • Recombinant Proteins
  • kaempferol
  • Genistein
  • Glycosyltransferases
  • biochanin A