Chlamydia trachomatisCT771 (nudH) Is an Asymmetric Ap₄A Hydrolase

Abstract

Asymmetric diadenosine 5′,5′″-P1,P4-tetraphosphate (Ap4A) hydrolases are members of the Nudix superfamily that asymmetrically cleave the metabolite Ap4A into ATP and AMP while facilitating homeostasis. The obligate intracellular mammalian pathogen Chlamydia trachomatis possesses a single Nudix family protein, CT771. As pathogens that rely on a host for replication and dissemination typically have one or zero Nudix family proteins, this suggests that CT771 could be critical for chlamydial biology and pathogenesis. We identified orthologs to CT771 within environmental Chlamydiales that share active site residues suggesting a common function. Crystal structures of both apo- and ligand-bound CT771 were determined to 2.6 Å and 1.9 Å resolution, respectively. The structure of CT771 shows a αβα-sandwich motif with many conserved elements lining the putative Nudix active site. Numerous aspects of the ligand-bound CT771 structure mirror those observed in the ligand-bound structure of the Ap4A hydrolase from Caenorhabditis elegans. These structures represent only the second Ap4A hydrolase enzyme member determined from eubacteria and suggest that mammalian and bacterial Ap4A hydrolases might be more similar than previously thought. The aforementioned structural similarities, in tandem with molecular docking, guided the enzymatic characterization of CT771. Together, these studies provide the molecular details for substrate binding and specificity, supporting the analysis that CT771 is an Ap4A hydrolase (nudH).

Metal ion coordination in the E. coli Nudix hydrolase dihydroneopterin triphosphate pyrophosphatase: New clues into catalytic mechanism

“…The positions of our metals match those found in structural homologs Ap4A and RppH, which have been crystallized with four [ 21 , 25 ] and three [ 22 , 26 ] Mg 2+ ions in their active site ( Fig 2E ). M1-M3 is represented in all three structures; RppH does not have a modeled metal ion in position of our M4, the metal ion that does not come in contact with protein residues.…”

“…Even studies of orthologs of the same enzyme, such as Ap4A [21, 26, 29], do not converge, leaving many open questions regarding the extent of mechanistic conservation across the Nudix family. In contrast to most available structures of Nudix hydrolases, the position and identity of four activating transition metal ions were enabled by our use of anomalous scattering data from transition metal ions that mimic Mg 2+ .…”

“…Nudix enzymes use nucleophilic substitution to hydrolyze their respective substrates, but literature reports vary in the residues involved in catalysis, and number and roles of divalent cations [ 6 , 28 ]. Even studies of orthologs of the same enzyme, such as Ap4A [ 21 , 26 , 29 ], do not converge, leaving many open questions regarding the extent of mechanistic conservation across the Nudix family. In contrast to most available structures of Nudix hydrolases, the position and identity of four activating transition metal ions were enabled by our use of anomalous scattering data from transition metal ions that mimic Mg 2+ .…”

“…bacilliformis Ap4A [ 29 ] and structures solved subsequently from other organisms such as C . trachomatis CT771 [ 26 ] that favor the requirement for three metal ions, equivalent to M1-M3 in our structures. Combined with the challenges encountered in attempts to obtain a substrate-analog bound structure and an apparent low intrinsic binding affinity of metal to DHNTPase, we raise the possibility M4, is in fact relevant to DHNTPase and possibly other Nudix hydrolases by activating the water for catalysis as well as stabilizing the pyrophosphate leaving group.…”

“…These residues are 3.8 or 3.5 Å from the metal-bridged solvent in those structures, respectively, similar to the 3.6 Å measured for Glu59 and the bridging solvent similarly positioned in DHNTPase. Alternative Glu residues have been proposed as the base in Ap4A orthologs [ 26 , 29 ], but the case has also been made that a protein-derived general base is not necessary for divalent Nudix hydrolases given substantial pKa-lowering of the water by the metal ions in the cluster [ 34 ]. Indeed, on the basis of its level of sequence conservation and position in our structures in direct line with the apparent M2-M4-activated water and sulfur atom in the sulfate anion, deprotonation of the solvent in DHNTPase may not require a protein-based general base.…”