Trypanothione reductase of Trypanosoma congolense: gene isolation, primary sequence determination, and comparison to glutathione reductase.
The gene encoding trypanothione reductase, the redox disulfide-containing flavoenzyme that is unique to the parasitic trypanosomatids (Shames et al., 1986), has been isolated from the cattle pathogen Trypanosoma congolense. Library screening was carried out with inosine-containing oligonucleotide probes encoding sequences determined from two active site peptides isolated from the purified Crithidia fasciculata enzyme. The nucleotide sequence of the gene was determined according to the dideoxy chain termination method of Sanger. The structural gene is 1476 nucleotides long and encodes 492 amino acids. We have identified the active site peptide containing the redox-active disulfide, a peptide corresponding to the histidine-467 region of human erythrocyte glutathione reductase, as well as the flavin binding domain that is highly conserved in all disulfide-containing flavoprotein reductase enzymes. Alignment of five tryptic peptides (80 residues) isolated from the C. fasciculata trypanothione reductase with the primary sequence of the T. congolense enzyme showed 88% homology with 76% identity. Additionally, a sequence comparison of the glutathione reductase from Escherichia coli or human erythrocytes to T. congolense trypanothione reductase reveals greater than 50% homology. A search for the amino acid residues in the primary sequence of trypanothione reductase functionally active in binding/catalysis in human erythrocyte glutathione reductase shows that only the two arginine residues (Arg-37 and Arg-347), shown by X-ray crystallographic data to hydrogen bond to the GS1 glutathione glycyl carboxylate, are absent.
Shames
S L
SL
Department of Chemistry, Biology, Massachusetts Institute of Technology, Cambridge 02139.
Kimmel
B E
BE
Peoples
O P
OP
Agabian
N
N
Walsh
C T
CT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005980:Glutathione Reductase / Q000032:analysis / Q000235:genetics*; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis / Q000235:genetics*; D014348:Trypanosoma congolense / Q000201:enzymology*
D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D005980:Glutathione Reductase