title: Evolution of the active site of human glutathione transferase A2-2 for enhanced activity with dietary isothiocyanates.
abstract: BACKGROUND
  Organic isothiocyanates (ITCs) are produced by plants, in which they are released from glucosinolates by myrosinase. ITCs are generally toxic and serve as a chemical defense against herbivorous insects and against infections by microorganisms. In mammalian tissues subtoxic concentrations of ITCs can provide protective effects against cancer and other diseases partially by induction of glutathione transferases (GSTs) and other detoxication enzymes. Thus, human consumption of edible plants rich in ITCs is presumed to provide health benefits. ITCs react with intracellular glutathione to form dithiocarbamates, catalyzed by GSTs. Formation of glutathione conjugates is central to the biotransformation of ITCs and leads to a route for their excretion. Clearly, the emergence of ITC conjugating activity in GSTs is essential from the biological and evolutionary perspective.
  
  
  METHODS
  In the present investigation an active-site-focused mutant library of GST A2-2 has been screened for enzyme variants with enhanced ITC activity.
  
  
  RESULTS
  Significantly superior activities were found in 34 of the approximately 2000 mutants analyzed, and the majority of the superior GSTs featured His and Gly residues in one of the three active-site positions subjected to mutagenesis.
  
  
  CONCLUSIONS
  We explored the propensity of GSTs to obtain altered substrate selectivity and moreover, identified a specific pattern of mutagenesis in GST for enhanced PEITC detoxification, which may play an important role in the evolution of adaptive responses in organisms subjected to ITCs.
  
  
  CONCLUSIONS
  The facile acquisition of enhanced ITC activity demonstrates that this important detoxication function can be promoted by numerous evolutionary trajectories in sequence space.
authors.affiliation: Department of Chemistry-BMC, Uppsala University, SE-75123 Uppsala, Sweden; Department of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 92037 La Jolla, CA, USA; Department Cell of Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 92037 La Jolla, CA, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Department of Cell & Molecular Biology, Computational & Systems Biology, Uppsala University, Sweden.
authors.forename: Daniel F A R
authors.initials: DF
authors.lastname: Dourado
authors.affiliation: Department of Chemistry-BMC, Uppsala University, SE-75123 Uppsala, Sweden; Department of Neurochemistry, Stockholm University, SE-10691 Stockholm, Sweden; Department of Chemical Physiology, The Scripps Research Institute, 92037 La Jolla, CA, USA. Electronic address: bengt.mannervik@neurochem.su.se.
authors.forename: Bengt
authors.initials: B
authors.lastname: Mannervik
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D004032:Diet; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D017879:Isothiocyanates / Q000494:pharmacology*; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D017879:Isothiocyanates; C037152:isothiocyanic acid; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha
keywords: Detoxication; Directed evolution; Enzyme engineering; Glutathione transferase; Isothiocyanate