Biochemical characterization of GAF domain of free-R-methionine sulfoxide reductase from Trypanosoma cruzi

Abstract

Trypanosoma cruzi is the causal agent of Chagas Disease and is a unicellular parasite that infects a wide variety of mammalian hosts. The parasite exhibits auxotrophy by L-Met; consequently, it must be acquired from the extracellular environment of the host, either mammalian or invertebrate. Methionine (Met) oxidation produces a racemic mixture (R and S forms) of methionine sulfoxide (MetSO). Reduction of L-MetSO (free or protein-bound) to L-Met is catalyzed by methionine sulfoxide reductases (MSRs). Bioinformatics analyses identified the coding sequence for a free-R-MSR (fRMSR) enzyme in the genome of T. cruzi Dm28c. Structurally, this enzyme is a modular protein with a putative N-terminal GAF domain linked to a C-terminal TIP41 motif. We performed detailed biochemical and kinetic characterization of the GAF domain of fRMSR in combination with mutant versions of specific cysteine residues, namely, Cys¹², Cys⁹⁸, Cys¹⁰⁸, and Cys¹³². The isolated recombinant GAF domain and full-length fRMSR exhibited specific catalytic activity for the reduction of free L-Met(R)SO (non-protein bound), using tryparedoxins as reducing partners. We demonstrated that this process involves two Cys residues, Cys⁹⁸ and Cys¹³². Cys¹³² is the essential catalytic residue on which a sulfenic acid intermediate is formed. Cys⁹⁸ is the resolutive Cys, which forms a disulfide bond with Cys¹³² as a catalytic step. Overall, our results provide new insights into redox metabolism in T. cruzi, contributing to previous knowledge of L-Met metabolism in this parasite.

Keywords: Free-R-methionine sulfoxide reductase; Methionine; Methionine sulfoxide; Redox metabolism; Trypanosoma cruzi.