Mutational analysis of parasite trypanothione reductase: acquisition of glutathione reductase activity in a triple mutant.
African trypanosomes contain a cyclic derivative of oxidized glutathione, N1,N8-bis(glutathionyl)spermidine, termed trypanothione. This is the substrate for the parasite enzyme trypanothione reductase, a key enzyme in disulfide/dithiol redox balance and a target enzyme for trypanocidal therapy. Trypanothione reductase from these and related trypanosomatid parasites is structurally homologous to host glutathione reductase but the two enzymes show mutually exclusive substrate specificities. To assess the basis of host vs parasite enzyme recognition for their disulfide substrates, the interaction of bound glutathione with active-site residues in human red cell glutathione reductase as defined by prior X-ray analysis was used as the starting point for mutagenesis of three residues in trypanothione reductase from Trypanosoma congolense, a cattle parasite. Mutation of three residues radically alters enzyme specificity and permits acquisition of glutathione reductase activity at levels 10(4) higher than in wild-type trypanothione reductase.
Sullivan
F X
FX
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Sobolov
S B
SB
Bradley
M
M
Walsh
C T
CT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003196:Computer Graphics; D005980:Glutathione Reductase / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D015345:Oligonucleotide Probes; D011487:Protein Conformation; D013379:Substrate Specificity; D014348:Trypanosoma congolense / Q000201:enzymology* / Q000235:genetics
D015345:Oligonucleotide Probes; D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D005980:Glutathione Reductase