Background. gamma-glutamylcysteine synthetase (GCS) catalyzes the first step in the biosynthesis of glutathione, which in trypanosome parasites is a required precursor to trypanothione. Essential by genetic and chemical data. An inhibitor of GCS, buthionine sulfoximine was shown to give partial cure of T. brucei infections in mice (1). This inhibitor is also effective at killing parasites in vitro. Knockdown of GCS by RNAi in procyclic T. brucei cells led to growth arrest and eventual cell death through the depletion of trypanothione (2). The growth arrest is rescued by supplementation of glutathione into the media. Structure and Assay. The enzyme can be expressed to high levels in bacteria (3-5). It can be readily assayed by a coupled spectrophotometric assay that would be amenable to HTS. X-ray structures from E. coli and plant have been reported (6-8). 1. B. Arrick, O. Griffith, A. Cerami, J. Exp. Med. 153, 720 (1981). 2. T. T. Huynh, V. T. Huynh, M. A. Harmon, M. A. Phillips, J. Biol. Chem. 278, 39794 (2003). 3. D. Lueder, M. Phillips, J. Biol. Chem. 271, 17485 (1996). 4. J. Abbott et al., J. Biol. Chem. 276, 42099 (2001). 5. J. Abbott, J. Ford, M. Phillips, Biochemistry 41, 2741 (2002). 6. M. Hothorn et al., J Biol Chem 281, 27557 (Sep 15, 2006). 7. T. Hibi et al., Proc Natl Acad Sci U S A 101, 15052 (Oct 19, 2004). 8. C. Lehmann et al., Proteins 56, 376 (Aug 1, 2004). Target ID: geneDB number = Tb10.389.1360 EC number (if enzyme) = EC 220.127.116.11 target name = Glutamate-cysteine ligase, or gamma-glutamylcysteine synthetase Assay: Assay exists but not yet in microtitre plate format Availability: Can be expressed > 1 mg/L in soluble, active form Activity: Small (< 1 full time person)
Target ID: geneDB number = Tb10.389.1360 EC number (if enzyme) = EC 18.104.22.168 target name = Glutamate-cysteine
Validation: Genetic AND chemical evidence
Assay status: Assay exists but not yet in microtitre plate format
Availability: Can be expressed > 1 mg/L in soluble, active form
Activity: Small (< 1 full time person)
Structure prediction and active site analysis of the metal binding determinants in gamma -glutamylcysteine synthetase. (2001).
J Biol Chem 276:
Substrate binding determinants of Trypanosoma brucei gamma-glutamylcysteine synthetase. (2002).
Gene knockdown of gamma-glutamylcysteine synthetase by RNAi in the parasitic protozoa Trypanosoma brucei demonstrates that it is an essential enzyme. (2003).
J Biol Chem 278:
YbdK is a carboxylate-amine ligase with a gamma-glutamyl:Cysteine ligase activity: crystal structure and enzymatic assays. (2004).
Crystal structure of gamma-glutamylcysteine synthetase: insights into the mechanism of catalysis by a key enzyme for glutathione homeostasis. (2004).
Proc Natl Acad Sci U S A 101:
Structural basis for the redox control of plant glutamate cysteine ligase. (2006).
J Biol Chem 281:
Inhibition of glutathione synthesis as a chemotherapeutic strategy for trypanosomiasis. (1981).
J Exp Med 153:
Characterization of Trypanosoma brucei gamma-glutamylcysteine synthetase, an essential enzyme in the biosynthesis of trypanothione (diglutathionylspermidine). (1996).
J Biol Chem 271: