Redox enzyme engineering: conversion of human glutathione reductase into a trypanothione reductase.

Abstract

The substrate specificity of the human enzyme glutathione reductase was changed from its natural substrate glutathione to trypanothione [N1,N8-bis(glutathionyl)spermidine] by site-directed mutagenesis of two residues. The glutathione analogue, trypanothione, is the natural substrate for trypanothione reductase, an enzyme found in trypanosomatids and leishmanias, the causative agents of diseases such as African sleeping sickness, Chagas disease, and Oriental sore. The rational bases for our mutational experiments were the availability of a high-resolution X-ray structure for human glutathione reductase with bound substrates, the active site sequence comparisons of human glutathione reductase and the trypanothione reductases from Trypanosoma congolense and Trypanosoma cruzi, a complementary set of mutants in T. congolense trypanothione reductase, and the properties of substrate analogues of trypanothione. Mutation of two residues, A34----E34 and R37----W37, in the glutathione-binding site of human glutathione reductase switches human glutathione reductase into a trypanothione reductase with a preference for trypanothione over glutathione by a factor of 700 using kcat/Km as a criterion.

• Authors: M Bradley, U S Bücheler, C T Walsh
• Journal: Biochemistry (Biochemistry) Vol. 30 Issue 25 Pg. 6124-7 (Jun 25 1991) ISSN: 0006-2960 [Print] United States
• PMID: 2059620 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• trypanothione
• NADH, NADPH Oxidoreductases
• trypanothione reductase
• Glutathione Reductase
• Glutathione
• Spermidine

Topics

• Animals
• Base Sequence
• Binding Sites
• Cloning, Molecular
• Escherichia coli (genetics)
• Glutathione (analogs & derivatives, metabolism)
• Glutathione Reductase (chemistry, genetics)
• Humans
• Kinetics
• Molecular Sequence Data
• Mutagenesis, Site-Directed
• NADH, NADPH Oxidoreductases (chemistry, genetics)
• Oxidation-Reduction
• Protein Engineering (methods)
• Spermidine (analogs & derivatives, metabolism)
• Substrate Specificity
• Trypanosoma (enzymology)
• X-Ray Diffraction