Post-translational modifications to catalytic cysteine by selenium enhance the enzyme activity of glyceraldehyde-3-phosphate dehydrogenase from Brassica oleracea var. italica.

Abstract

Selenium (Se) was recently shown to be beneficial for plants, and its application to crop production, drug development, and environmental hygiene is expected. The enzyme activities of some proteins were previously reported to be enhanced in plants grown with Se. These increases may be attributed to the binding of Se to catalytic cysteine by post-translational modifications (PTMs), which is a novel mechanism of expression. Therefore, the present study investigated Se binding and increases in enzyme activity by PTMs to cytoplasmic glyceraldehyde-3-phosphate dehydrogenase (GAPC) from broccoli (B. oleracea var. italica), which is classified as a Se-accumulating species, in planta and in vitro. GAPC derived from plants cultivated with 1 µM selenate had more Se bonds and stronger enzyme activity than that from those cultivated without selenate. BES-Thio, a fluorescent probe that identifies thiol or selenol groups, revealed that increases in GAPC activity by Se binding were due to the formation of a selenol group, which is more reactive than a thiol group, on GAPC-catalyzed cysteine by PTMs. Furthermore, purified recombinant GAPC and mutants (C156S, C160S, and C156S/C160S) were reduced and reacted with GSSeSG in vitro to investigate Se binding and selenol group generation by PTMs to recombinant GAPC and mutant proteins. The results obtained show that Se binding and selenol group generation by PTMs occurred only at Cys 156, which corresponds to the catalytic Cys. In addition, V_max, K_cat, and K_cat /K_m values of Se binding GAPC synthesized in vitro using purified BoGAPC were 1.49-, 1.48-, and 1.86-fold higher, respectively, than those of GAPC. These results indicate that the generation of selenol group at the catalytic Cys of GAPC by PTM improves the enzyme activity.

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