Glyoxalase II of African trypanosomes is trypanothione-dependent

J Biol Chem. 2004 May 21;279(21):22209-17. doi: 10.1074/jbc.M401240200. Epub 2004 Feb 19.

Abstract

The glyoxalase system is a ubiquitous pathway catalyzing the glutathione-dependent detoxication of ketoaldehydes such as methylglyoxal, which is mainly formed as a by-product of glycolysis. The gene encoding a glyoxalase II has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness. The deduced protein sequence contains the highly conserved metal binding motif THXHXDH but lacks three basic residues shown to fix the glutathione-thioester substrate in the crystal structure of human glyoxalase II. Recombinant T. brucei glyoxalase II hydrolyzes lactoylglutathione, but does not show saturation kinetics up to 5 mm with the classical substrate of glyoxalases II. Instead, the parasite enzyme strongly prefers thioesters of trypanothione (bis(glutathionyl)spermidine), which were prepared from methylglyoxal and trypanothione and analyzed by high performance liquid chromatography and mass spectrometry. Mono-(lactoyl)trypanothione and bis-(lactoyl)trypanothione are hydrolyzed by T. brucei glyoxalase II with k(cat)/K(m) values of 5 x 10(5) m(-1) s(-1) and 7 x 10(5) m(-1) s(-1), respectively, yielding d-lactate and regenerating trypanothione. Glyoxalase II occurs in the mammalian bloodstream and insect procyclic form of T. brucei and is the first glyoxalase II of the order of Kinetoplastida characterized so far. Our results show that the glyoxalase system is another pathway in which the nearly ubiquitous glutathione is replaced by the unique trypanothione in trypanosomatids.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Blotting, Western
  • Chromatography, Gel
  • Chromatography, High Pressure Liquid
  • Cloning, Molecular
  • Dose-Response Relationship, Drug
  • Glutathione / analogs & derivatives*
  • Glutathione / chemistry*
  • Glycolysis
  • Humans
  • Hydrogen-Ion Concentration
  • Ions
  • Kinetics
  • Lactic Acid / chemistry
  • Mass Spectrometry
  • Metals / chemistry
  • Models, Chemical
  • Molecular Sequence Data
  • Recombinant Proteins
  • Sequence Homology, Amino Acid
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Spermidine / analogs & derivatives*
  • Spermidine / chemistry*
  • Thiolester Hydrolases / chemistry*
  • Thiolester Hydrolases / genetics
  • Time Factors
  • Trypanosoma brucei brucei

Substances

  • Ions
  • Metals
  • Recombinant Proteins
  • Lactic Acid
  • trypanothione
  • Thiolester Hydrolases
  • hydroxyacylglutathione hydrolase
  • Glutathione
  • S-lactoylglutathione
  • Spermidine

Associated data

  • GENBANK/AJ492819