Characterization of Trypanosoma brucei γ-Glutamylcysteine Synthetase, an Essential Enzyme in the Biosynthesis of Trypanothione (Diglutathionylspermidine)

Deirdre V Lueder,Margaret A Phillips

doi:10.1074/jbc.271.29.17485

Abstract

The parasitic protozoan Trypanosoma brucei maintains redox balance by synthesizing a conjugate of glutathione and spermidine termed trypanothione. The first committed step in the biosynthesis of glutathione, and thereby trypanothione, is catalyzed by the enzyme gamma-glutamylcysteine synthetase (gammaGCS). We have cloned and sequenced the 2037-base pair gene coding for the catalytic subunit of T. brucei gammaGCS. T. brucei gammaGCS appears to be encoded by a single copy gene. A transcript of about 2.3 kilobases was observed in procyclic trypomastigotes. The deduced amino acid sequence of 679 amino acids shares 45, 41, and 36% sequence identity with mammalian, Caenorhabditis elegans, and yeast gammaGCS, respectively. The T. brucei gammaGCS gene was expressed in E. coli; the purified 77.4-kDa enzyme catalyzes the ligation of L-Glu to L-Cys with a kcat of 10 s-1, confirming that the gene encodes the functional catalytic subunit of gammaGCS. The apparent Km values measured for the three natural substrates L-Glu, L-Cys, and ATP are 0.24, 0.69, and 0.07 mM, respectively. Unlike the mammalian enzyme, L-alpha-aminobutyrate (apparent Km = 10 mM) is a poor substitute for L-Cys in the T. brucei gammaGCS-catalyzed reaction. T. brucei gammaGCS is feedback-inhibited by glutathione (apparent KI = 1.1 mM), and it is inactivated by cystamine and buthionine sulfoximine. The kinetic properties of recombinant T. brucei gammaGCS suggest that the substrate binding pocket and the mechanism of enzyme regulation differ from the mammalian enzyme, providing evidence that T. brucei gammaGCS could be a selective chemotherapeutic target for the treatment of trypanosomiasis.

Highlights

Pressed in E. coli; the purified 77.4-kDa enzyme catalyzes the ligation of L-Glu to L-Cys with a kcat of 10 s؊1, confirming that the gene encodes the functional catalytic subunit of ␥GCS
T. brucei ␥GCS is feedback-inhibited by glutathione, and it is inactivated by cystamine and buthionine sulfoximine
Isolation and Characterization of Clones—Degenerate oligonucleotide primers were designed to two amino acid sequence regions conserved between the rat kidney catalytic subunit [34] and S. cerevisiae ␥GCS [14], corresponding to the motif MGFGMG and to HFENIQSTNWQT

Summary

Introduction

Pressed in E. coli; the purified 77.4-kDa enzyme catalyzes the ligation of L-Glu to L-Cys with a kcat of 10 s؊1, confirming that the gene encodes the functional catalytic subunit of ␥GCS. A full-length clone of the T. brucei ␥GCS gene was obtained from a genomic library and encodes a 679-amino acid protein (molecular mass ϭ 77.4 kDa) sharing 45, 41, and 36% sequence identity with the mammalian catalytic subunit, C. elegans, and yeast ␥GCS, respectively.

Results

Conclusion