SITE-DIRECTED MUTAGENESIS OF THE REDOX-ACTIVE CYSTEINES OF TRYPANOSOMA-CRUZI TRYPANOTHIONE REDUCTASE

The gene for trypanothione reductase from the Silvio strain of Trypano soma cruzi has been cloned, sequenced and overexpressed in Escherichia coli using the constitutive Ipp promoter on the expression plasmid pB STNAV. Up to 13% of the total soluble protein is enzymically active tr ypanothione reductase with kinetic properties similar to the enzyme pu rified from T. cruzi. In order to assess the catalytic role of the put ative active-site cysteine residues (C53 and C58), three mutant protei ns have been constructed by site-directed mutagenesis substituting ala nine or serine residues for cysteine; [C53A]trypanothione reductase, [ C53S]trypanothione reductase and [C58S]trypanothione reductase. Althou gh the purified, recombinant mutant proteins were catalytically inacti ve with NADPH and trypanothione disulphide as substrates, all showed c omparable levels of transhydrogenase activity between NADPH and thio-N ADP(+), suggesting that the mutant proteins had correctly folded in vi vo. All three mutants showed substantially different catalytic paramet ers for thio-NADP(+) than the wild-type enzyme, presumably as a conseq uence of modifying the environment of the enzyme-bound flavin, thereby altering its chemical reactivity. The purified [C58S]trypanothione re ductase showed spectral properties similar to the oxidised wild-type e nzyme but, unlike the wild-type enzyme, did not acquire the characteri stic charge-transfer complex of the EH, form on addition of NADPH. Tn contrast, in the absence of NADPH both [C53A]trypanothione reductase a nd [C53S]trypanothione reductase showed spectral properties similar to the EH, form of the wild-type enzyme. These data indicate that both C 53 and C58 are essential for overall catalysis, with the thiolate anio n of C58 interacting with the enzyme-bound FAD and C53 interacting wit h the disulphide substrate. These mutants should be useful in crystall ographic studies of reaction intermediates which cannot be obtained wi th the catalytically active native enzyme.