Enzymatic properties of Myxococcus xanthus exopolyphosphatases mxPpx1 and mxPpx2

Myxococcus xanthus possesses two exopolyphosphatases, mxPpx1 and mxPpx2, which belong to the family of Ppx/GppA phosphatases; however, their catalytic properties have not been described. mxPpx1 and mxPpx2 contain 311 and 505 amino acid residues, respectively; mxPpx2 has an additional C-terminal region, which corresponds to the metal-dependent HDc phosphohydrolase domain. mxPpx1 mainly hydrolyzed short-chain polyPs (polyP₃ and polyP₄), whereas mxPpx2 preferred long-chain polyP_60-70 and polyP_700-1000. mxPpx2 was activated by 25-50 mM KCl, but mxPpx1 did not significantly depend on K⁺. In addition, mxPpx1 and mxPpx2 showed weak hydrolysis of ATP and GTP in the absence of K⁺, and mxPpx2 could also hydrolyze guanosine pentaphosphate (pppGpp) in the presence of K⁺. The exopolyphosphatase activity of mxPpx1 toward polyP₃ was inhibited by polyP_700-1000 and that of mxPpx2 toward polyP_60-70 and polyP_700-1000, by pyrophosphate. To clarify the function of the mxPpx2 C-terminal domain, it was fused to mxPpx1 (mxPpx1-2C) and deleted from mxPpx2 (mxPpx2∆C). Compared to wild-type mxPpx2, mxPpx2∆C had significantly reduced exopolyphosphatase activity toward long-chain polyPs (by 90%), whereas that toward polyP₃ and polyP₄ was much less affected; furthermore, the phosphohydrolase activity toward pppGpp, ATP, and GTP was also decreased (by 30-75%). In contrast, mxPpx1-2C had increased hydrolytic activity compared to mxPpx1. Furthermore, mxPpx2∆C lost the requirement for K⁺ characteristic for the wild-type enzyme, whereas mxPpx1-2C acquired it. These results suggest that the C-terminal domain of mxPpx2 is necessary for its maximum hydrolytic activity, especially toward long-chain polyPs, and defines mxPpx2 dependency on K⁺ for activation.