Role of metal cations and oxyanions in the regulation of protein arginine phosphatase activity of Yw1E from Bacillus subtilis

Protein arginine phosphorylation (pArg) is a relatively novel posttranslational modification. Protein arginine phosphatase Yw1E negatively regulates arginine phosphorylation and consequently induces the expression of stress-response genes that are crucial for bacterial stress tolerance and pathogenic homolog Staphylococcus aureus virulence. However, little is known about the factors that affect the enzymatic activity of Yw1E with the exception of the effect of oxidative stress. Herein, based on the hydrolysis of the chromogenic substrate p-nitrophenyl phosphate (pNPP) by Yw1E, we investigate the role of metal cations and oxyanions in the regulation of Yw1E activity. Interestingly, among the various cations that we tested, Ca2+ activates Yw1E, while other cations, including Ag+, Co2+, Cd2+, and Zn2+, are inhibitory. Furthermore, as chemical analogues of phosphate, oxyanions play multiple roles in phosphatase activity. The regulatory switch Cys within the catalytic site regulates Yw1E activity. Specifically, the thiol of this Cys could be alkylated by IAM (iodoacetamide) or oxidized by H2O2, resulting in enzymatic inhibition. Conversely, reducing reagents, such as DTT (dithiothreitol), beta-me (beta-mercaptoethanol), and TCEP (tris(2-carboxyethyl)phosphine) enhance Yw1E activity. Additionally, as a stable analogue to pArg, pAIE binds to Yw1E with a K-d of 149.1 nM and a binding stoichiometry n of 1.2 and inhibits Yw1E with an IC50 of 316.3 +/- 12.73 mu M. The inhibition and activation of Yw1E may have broad implications for the physiology, pharmacology and toxicology of metal cations and oxyanions.