LAUSR

Cyclic dinucleotides are signaling molecules that modulate many processes, including immune response and virulence factor production. Their cellular levels in bacteria are fine-tuned by metal-dependent phosphodiesterases, namely the EAL and HD-GYP proteins, with HD-GYPs belonging to the larger HD-domain superfamily. In the present study, we first focus on the catalytic properties, range of metal ions and substrates of the HD-[HD-GYP] subfamily, consisting of two HD-domains. We identified SO3491 as a homolog of the VCA0681 and the second example of an HD-[HD-GYP]. Both proteins hydrolyze c-di-GMP and 3'3'c-GAMP and coordinate various metal ions, but only Fe and to a lesser extent Co support hydrolysis. The proteins are active only in the di-ferrous form and not in the one-electron more oxidized FeIIFeIII state. Although the C-terminal HD-GYP domain is essential for activity, the role of the N-terminal HD-domain remains unknown. We show that the N-terminal site is essential for protein stability, influences the individual apparent kcat and KM (but not kcat/KM), and does not coordinate substrates thus precluding its involvement in cyclic dinucleotide sensing. We proceeded to perform phylogenetic analyses so as to examine the distribution and functional relationships of the HD-[HD-GYP]s to the rest of HD-GYPs. The phylogeny provides a correlation map that draws a link between the evolutionary and functional diversification of HD-GYPs, serving as a template for predicting the chemical nature of the metallocofactor, level of activity, and reaction outcome.