Mutational analysis of parasite trypanothione reductase: acquisition of glutathione reductase activity in a triple mutant

• Published in: Biochemistry (Easton) Vol. 30; no. 11; pp. 2761 - 2767
• Main Authors: Sullivan, Francis X, Sobolov, Susan B, Bradley, Mark, Walsh, Christopher T
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.03.1991
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Biological and medical sciences; Computer Graphics; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Glutathione Reductase - genetics; Glutathione Reductase - metabolism; glutathionine reductase; Kinetics; Models, Molecular; Molecular Sequence Data; mutagenesis; Mutagenesis, Site-Directed; NADH, NADPH Oxidoreductases - genetics; NADH, NADPH Oxidoreductases - metabolism; Oligonucleotide Probes; Oxidoreductases; Protein Conformation; Substrate Specificity; Trypanosoma congolense - enzymology; Trypanosoma congolense - genetics; trypanothione reductase; Protozoa; Host parasite relation; Enzymatic activity; Enzyme; Substrate specificity; Parasiticid; Trypanosoma congolense; Rhizoflagellata; Glutathione reductase (NAD(P)H); Kinetics; Mutation
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00225a004

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.