NMR reporters of stereospecific methionine sulfoxide reductase Activity

ANDRÉS BINOLFI; VERÓNICA A. LOMBARDO; FRANCO A. BIGLIONE; CAROLINA SÁNCHES-LOPÉZ; BRUNO MANTA PORTEIRO; NATALIA LABADIE

Buenos Aires

Taller; IV Taller de Resonacia Magnética; 2018

La Fundación del Instituto Leloir con el apoyo de CONICET

A reversible covalent modification in biological systems such as the oxidation of methionine (Met) side chains, is now emerging as a post-translational modification capable of regulating protein activity and cellular processes, such as the activation of Ca2+-calmodulin dependent protein kinase II or the remodeling of the actin cytoskeleton.1,2 Methionine side chain can be oxidized to the S- and R-diastereomers of methionine sulfoxide (MetOx). The reduction of MetOx is catalyzed by methionine sulfoxide reductases (MSRs). These enzymes include MSRA and MSRB, which specifically reduce MetOx S- and R-diastereomers, respectively. MetOx display unique NMR signals, including different sets of resonances for its S and R diastereomers that are readily identified in the NMR spectra.3 These changes of the NMR observables can be analyzed to describe MSRA and MSRB enzymatic activity in vitro using over-expressed or endogenous (from bacteria total protein extract) enzyme. In this work, we used free isotopically enriched L-MetOx and protein-bound MetOx as substrates and NMR reporters of stereospecific MSRA and MSRB activity. With this strategy, we validated that the catalytic efficiency (kcat/KM) for purified MSRA was 100-fold greater than MSRB for both reporters/substrates. On the other hand, the study in cell extracts showed that MSRB activity in bacteria increased towards the free amino acid. The design of new NMR reporters with stereospecific substrate recognition will be a powerful tool for the study of MSRs activity both in vitro and in vivo.