Recognition of ADPR by Bd-NDPSase.

<p><b>A)</b> Ribbon representation of the catalytic site of the ADPR-bound E140Q Bd-NDPSase crystal structure (PDB ID 5C7T). <b>B)</b> Schematic representation of the recognition of ADPR by Bd-NDPSase. Catalytic helix (α1) residues are shown in cyan, catalytic loop L9 residues are shown in magenta. N-terminal domain residues (1–44) are shown in green as is the specificity loop L7. Hydrogen bonds are shown as orange dashes. The prime symbol (‘) denotes residues of the opposite monomer.</p

Bd-NDPSase wild type and E140Q substrate specificity.

<p><b>A)</b> Wild type enzyme (gray bars) exhibited preference for nucleoside diphosphate sugar (NDPS). E140Q mutant (red bars) were catalytically inactive. <b>B)</b> Initial rates of GDPM hydrolysis for the wild type and E140Q mutant were fit by nonlinear least squares to the Michaelis-Menten equation (solid lines) to determine k_cat (5.2 (ms)^-1) and K_m (0.3 mM). Standard deviations of triplicate measurements are shown by the shaded bars for the wild type (gray shade) and mutant (red shade).</p

Cellular localization of Bd-NDPSase.

<p><b>A-B)</b> TEM micrographs at 37,000x magnification showing the location of the secondary gold antibody (black dots) in the periplasmic space of a <i>Bdellovibrio bacteriovorus</i> cell. <b>C)</b> TEM micrographs at 20,000x magnification showing the location of the secondary gold antibody (solid, black spots) in the periplasmic space of multiple <i>Bdellovibrio bacteriovorus</i> cells.</p

Sugar recognition by bacterial Nudix sugar hydrolases.

<p><b>A)</b> Ribose recognition by the Bd-NDPSase (PDB ID 5C7T). <b>B)</b> Mannose recognition by the Ec-NDPS hydrolase (PDB ID 3O61). <b>C)</b> Ribose recognition by the Ec-ADPR hydrolase (PDB ID 1KHZ). Substrate carbons are shown in black, residue carbons are colored using the main chain color convention. Nitrogen and Oxygen are colored in blue and red respectively. Hydrogen bonds are shown as orange dashes. The prime symbol (‘) denotes residues of the opposite monomer.</p

Overall Structure Bd-NDPSase.

<p><b>A)</b> Structure of E140Q Bd-NDPSase bound to ADPR (PDB ID 5C7T). <b>B)</b> Sequence alignment of the Ec-ADPRase (PDB ID 1KHZ) and Ec-NDPSase (PDB ID 3O61) to Bd-NDPSase. The N-terminal domain (residues 1–44) consists of an antiparallel beta sheet (β1- β3) and is denoted by green. The Nudix fold consists of a mixed beta sheet (β1- β10, yellow) flanked by helix α1 (cyan) on one side and helices α2 and α3 (residues 148–182, gray) on the other side. The location of the E140Q mutation on loop L9 (magenta) is denoted by an asterisk. The prime symbol (‘) denotes residues of the opposite monomer (lighter color shade).</p

Nucleoside recognition by bacterial Nudix sugar hydrolases.

<p>In all cases, the nucleoside is stacked by an aromatic residues in loop L1 and an arginine in strand β3’. <b>A)</b> Adenosine recognition by the Bd-NDPSase hydrolase (PDB ID 5C7T). <b>B)</b> Guanosine recognition by the Ec-NDPSase hydrolase (PDB ID 3O61). <b>C)</b> Adenosine recognition by the Ec-ADPR hydrolase (PDB ID 1KHZ). Substrate carbons are shown in black, residue carbons are colored using the main chain color convention. Nitrogen and Oxygen are colored in blue and red respectively. Hydrogen bonds are shown as orange dashes. The prime symbol (‘) denotes elements of the opposite monomer.</p

Structural and Enzymatic Characterization of a Nucleoside Diphosphate Sugar Hydrolase from <i>Bdellovibrio bacteriovorus</i>

<div><p>Given the broad range of substrates hydrolyzed by Nudix (<i>nu</i>cleoside <i>di</i>phosphate linked to <i>X</i>) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 –- a Nudix hydrolase from <i>Bdellovibrio bacteriovorus–</i>that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively). Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-β-α NDPSase fold differentiates NDPSases from ADPRases.</p></div

Data collection and refinement statistics for Bd-NDPSase.

<p>Numbers in parenthesis show the values for the highest resolution shell</p><p>Data collection and refinement statistics for Bd-NDPSase.</p