Conversion of hydroxyphenylpyruvate dioxygenases into hydroxymandelate synthases by directed evolution

FEBS Lett. 2006 Jun 12;580(14):3445-50. doi: 10.1016/j.febslet.2006.05.018. Epub 2006 May 15.

Abstract

Hydroxymandelate synthase (HmaS) and hydroxyphenylpyruvate dioxygenase (HppD) are non-heme iron-dependent dioxygenases, which share a common substrate and first catalytic step. The catalytic pathways then diverge to yield hydroxymandelate for secondary metabolism, or homogentisate in tyrosine catabolism. To probe the differences between these related active sites that channel a common intermediate down alternative pathways, we attempted to interconvert their activities by directed evolution. HmaS activity was readily introduced to HppD by just two amino acid changes. A parallel attempt to engineer HppD activity in HmaS was unsuccessful, suggesting that homogentisate synthesis places greater chemical and steric demands on the active site.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalysis
  • Dioxygenases / chemistry
  • Dioxygenases / metabolism*
  • Directed Molecular Evolution*
  • Ligases / metabolism*
  • Models, Molecular
  • Plasmids

Substances

  • Dioxygenases
  • Ligases