HD-[HD-GYP] Phosphodiesterases: Activities and Evolutionary Diversification within the HD-GYP Family

Abstract

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 this study, we first focus on the catalytic properties and the range of metal ions and substrates of the HD-[HD-GYP] subfamily, consisting of two HD domains. We identified SO3491 as a homologue of 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 diferrous form and not in the one-electron more oxidized Fe II Fe III 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 important for protein stability, influences the individual apparent k cat and K M (but not k cat /K M ), and cannot bind c-di-GMP, thus precluding its involvement in cyclic dinucleotide sensing. We proceeded to perform phylogenetic analyses to examine the distribution and functional relationships of the HD-[HD-GYP]s to the rest of the 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.

The HD-Domain Metalloprotein Superfamily: An Apparent Common Protein Scaffold with Diverse Chemistries

“…Hydrolases are further subdivided into (i) phosphatases, including dGTPases [38], RelA/SpoT [9,17], SAMHD1 [18,19], EF1143 [20,21], etc. and (ii) phosphodiesterases (PDEs), including exoribonucleases [39], PDEases [27][28][29][30][31][32][33][34], Cas proteins [23][24][25][26], and HD-GYPs [14,35,36,40] (Figure 1B). Phosphatases hydrolyze a multitude of (deoxy)nucleotide-based substrates that vary in the identity of their base(s) and the extent of phosphorylation (Figure 2) [3,5,16,22].…”

“…However, because single amino acid substitutions of each of the GYP domain residues to alanines hardly affect PDE activity [36], its role in catalysis and protein stability remains poorly understood. The GYP motif is considered important for interaction with the GGDEF cyclase (named after its highly conserved Gly-Gly-Asp-Glu-Phe sequence motif) [8] and serves as a substrate specificity element for the recognition of c-di-GMP and its hybrid 3 3 -cGAMP analog [40].…”

“…Dinuclear HD-GYPs can only perform a one-step hydrolysis, whereas trinuclear ones can perform a two-step hydrolysis, leading to the respective monophosphates. Metal ions that can stimulate hydrolysis are Fe, Mn, and, to a lesser extent, Co and Ni [40].…”

“…Unlike other dinuclear nonheme-iron oxygenases, which utilize the fully reduced Fe II /Fe II form of their cofactors, HD-domain oxygenases stabilize a mixedvalent Fe II /Fe III state for the four-electron oxidation of a C-C/P bond and do not require an external reducing system for reactivation of the cofactor (i.e., after one substrate turnover the site returns to the Fe II -Fe III form) [15]. Stabilization under the same redox conditions of the Fe II Fe III cofactor in oxygenases and the Fe II Fe II cofactor in HD-domain hydrolases suggests that the HD-domain sites may tune activity through the modulation of cofactor reduction potentials [40]. PhnZ and TmpB were later established as oxygenases, demonstrating that MIOX is not a functional outlier [12,13].…”

Sequence conservation, domain architectures, and phylogenetic distribution of the HD-GYP type c-di-GMP phosphodiesterases

“…These include the partially conserved Glu residue in the N-terminal loop (E185 in the structure of P. marina PmGH, PDB ID: 4MCW, Figure 1). This acidic residue (Glu or Asp) participates in binding the third metal atom; phylogenetic analyses showed that, depending on the presence or absence of this Glu residue, HD-GYP domains could be split into two families carrying, respectively, trinuclear or binuclear metal centers (45,46,58). For their c-di-GMP phosphodiesterase activity, experimentally studied HD-GYP domains typically require two ferrous (Fe 2+ ) ions, oxidation of either one of them inactivates the enzyme (36).…”