Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

Abstract

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

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

“…However, SO3491 (and VCA0681) exhibits an almost 3 order of magnitude lower k cat / K M for 3′3′c-GAMP than for c-di-GMP. This finding is to a certain degree unexpected, because (i) VCA0681 is purported to be the primary regulator of 3′3′c-GAMP V. cholerae and (ii) Vc EAL, a 3′3′c-GAMP specific EAL domain PDE, exhibits the same catalytic efficiency for both c-di-GMP and 3′3′c-GAMP . At present, it is unclear whether the differential activity for the two substrates is specific to HD-[HD-GYP]­s.…”

“…HD-[HD-GYP]s and EALs are shown to be catalytically similar and exhibit comparable k cat /K M values for c-di-GMP. [47][48][49][50]53 The apparent K M s of SO3491 (and VCA0681) can be well matched to the cellular levels of c-di-GMP in V. cholerae, which are reported to be 0.9−20 μM. 59 However, SO3491 (and VCA0681) 60 exhibits an almost 3 order of magnitude lower k cat /K M for 3′3′c-GAMP than for c-di-GMP.…”

Regulation of Exopolysaccharide Production by ProE, a Cyclic-Di-GMP Phosphodiesterase in Pseudomonas aeruginosa PAO1

“…It has been well documented that different cations may generate variable impact on enzyme structure and activity. Similarly, it was shown recently that another two PDEs, CnpB from Mycobacterium tuberculosis and VcEAL (VC0395_A1247) from Vibrio cholerae also showed the best performance with Co 2+ (Yang et al, 2014;Yadav et al, 2019). It is likely that ProE may share a similar metal-ion-dependent catalysis mechanism with these enzymes.…”

“…Nevertheless, cation ion seems only affect catalysis but doesn't influence the enzyme substrate specificity. ProE degraded only c-di-GMP ( Supplementary Figure S8), whereas CnpB and VcEAL could degrade not only c-di-GMP, but also c-di-AMP or cGAMP, respectively (Yang et al, 2014;Yadav et al, 2019).…”

A pGpG-specific phosphodiesterase regulates cyclic di-GMP signaling in Vibrio cholerae

“…2 F ) that can be produced following the degradation of 3ʹ, 3ʹ-cGAMP ( 36 ). Several enzymes have been reported to exhibit bifunctional activity that produces or degrades both c-di-GMP and cGAMP ( 37 , 38 , 39 ), suggesting that the concentration of two cyclic dinucleotides are interactively regulated. In this context, it is probable that PggH could act as a mediator in the cross talk between the c-di-GMP and cGAMP signaling pathway.…”