Altering UDP-glucose Donor Substrate Specificity of Bacillus licheniformis Glycosyltransferase towards TDP-glucose

J Microbiol Biotechnol. 2019 Feb 28;29(2):268-273. doi: 10.4014/jmb.1811.11009.

Abstract

The specificity of a Bacillus licheniformis uridine diphosphate (UDP) glycosyltransferase, YjiC, was increased towards thymidine diphosphate (TDP)-sugar by site-directed mutagenesis. The Arg-282 of YjiC was identified and investigated by substituting with Trp. Conversion rate and kinetic parameters were compared between YjiC and its variants with several acceptor substrates such as 7-hydroxyflavone (7-HF), 4',7-dihydroxyisoflavone, 7,8-dihydroxyflavone and curcumin. Molecular docking of TDP-glucose and 7-HF with YjiC model showed pi-alkyl interaction with Arg-282 and His-14, and pi-pi interaction with His14 and thymine ring. YjiC (H14A) variant lost its glucosylation activity with TDP-glucose validating significance of His-14 in binding of TDP-sugars.

Keywords: Glycosyltransferase; TDP-glucose substrate specificity; enzyme Kinetics; mutation; protein engineering.

MeSH terms

  • Amino Acid Sequence
  • Bacillus licheniformis / enzymology*
  • Bacillus licheniformis / genetics
  • Binding Sites / genetics
  • Flavonoids / metabolism
  • Glycosylation
  • Glycosyltransferases / chemistry*
  • Glycosyltransferases / genetics
  • Glycosyltransferases / metabolism*
  • Kinetics
  • Molecular Docking Simulation
  • Mutation
  • Nucleoside Diphosphate Sugars / metabolism*
  • Protein Engineering
  • Structure-Activity Relationship
  • Substrate Specificity / genetics
  • Uridine Diphosphate Glucose / metabolism*

Substances

  • Flavonoids
  • Nucleoside Diphosphate Sugars
  • thymidine diphosphate sugars
  • Glycosyltransferases
  • Uridine Diphosphate Glucose
  • 7-hydroxyflavone