Engineering the substrate specificity of glutathione reductase toward that of trypanothione reduction.

摘要: Abstract Glutathione reductase (EC 1.6.4.2; CAS registry number 9001-48-3) and trypanothione (CAS 102210-35-5), which are related flavoprotein disulfide oxidoreductases, have marked specificities for glutathione trypanothione, respectively. A combination of primary sequence alignments molecular modeling, together with the high-resolution crystal structure human reductase, identified certain residues as potentially being responsible substrate discrimination. Site-directed mutagenesis Escherichia coli was used to test these predictions. The mutation Asn-21 Arg demonstrated that this single change insufficient generate greater discrimination against shown by compared E. enzyme. However, Ala-18, Asn-21, Arg-22 amino acid (Glu, Trp, Asn, respectively) in corresponding positions Trypanosoma congolense confirmed region polypeptide chain is intimately involved recognition. It led a mutant form possessed essentially no activity but able catalyze reduction kcat/Km value 10% measured natural reductases. These results should be considerable importance design trypanocidal drugs targeted at differences between metabolism trypanosomatids their hosts.