Structure of a coenzyme A pyrophosphatase from Deinococcus radiodurans: a member of the Nudix family

Abstract

Gene Dr1184 from Deinococcus radiodurans codes for a Nudix enzyme (DR-CoAse) that hydrolyzes the pyrophosphate moiety of coenzyme A (CoA). Nudix enzymes with the same specificity have been found in yeast, humans, and mice. The three-dimensional structure of DR-CoAse, the first of a Nudix hydrolase with this specificity, reveals that this enzyme contains, in addition to the fold observed in other Nudix enzymes, insertions that are characteristic of a CoA-hydrolyzing Nudix subfamily. The structure of the complex of the enzyme with Mg(2+), its activating cation, reveals the position of the catalytic site. A helix, part of the N-terminal insertion, partially occludes the binding site and has to change its position to permit substrate binding. Comparison of the structure of DR-CoAse to those of other Nudix enzymes, together with the location in the structure of the sequence characteristic of CoAses, suggests a mode of binding of the substrate to the enzyme that is compatible with all available data.

FIG. 1.

Alignment of the sequences of characterized Nudix CoAses. Conserved residues are shown as white letters in black boxes. Conservative substitutions are shown with grey letters in open boxes. Stars indicate residues of the UPF0035 (NuCoA) motif; triangles indicate residues of the Nudix motif. GenBank GI numbers for sequences are as follows: DR1184, D. radiodurans, gi 15806203; Nudt7_H, human, gi 1848966; Nudt7_M, mouse, gi 12746409; Pcdlp, yeast, gi 6323180.

FIG. 2.

Structure of the DR-CoAse. (A) Schematic representation of the three-dimensional structure of DR-CoAse. The regions corresponding to the Nudix fold are magenta, those of NuCoA are turquoise (side chains in dark blue), and the rest of the structure is emerald green. The side chains of the Nudix motif are shown in orange. (B) Secondary structure diagram of the DR-CoAse. Strands are shown as arrows, helices are shown as cylinders, and connecting regions are shown as black lines. Dashed lines show regions that were not built in the structure. The beginning and the end of each element are shown.

FIG. 3.

Electron density of the coordination of the Mg²⁺ ion. The Mg²⁺ ion has a near-perfect octahedral coordination formed by five water molecules and the side chain of Glu 86 of the Nudix motif. Other side chains surrounding the position of the ion are also shown.

FIG. 4.

Comparison of the Nudix hydrolases of known structure. (A) Sequence comparison based on the alignment of the three-dimensional structure of the four enzymes. No attempt was made to align the sequences of the regions not structurally conserved (black letters). Secondary structure elements are indicated: helices with thick lines, strands with arrows. Elements common to all four enzymes are shown in red. Elements characteristic of individual enzymes are shown in blue. Bold characters show the helices. (B, C, D, and E) Schematic representation of the four known structures of Nudix hydrolases. The Nudix fold (magenta), including the additional three-stranded sheet (pink), is highlighted in all the structures. (B) MutT. This enzyme consists of only these elements. (C) EC-ADPRase. Additional portions of the structure, including the N-terminal extension involved in dimer formation by domain swapping, are shown in turquoise. (D) Ap4Ase. Additional regions, including the α-helical insertion, are shown in orange. (E) DR-CoAse. Additional regions, including the N-terminal and the C-terminal extensions, are shown in green.

FIG. 5.

Binding of CoA to DR-CoAse. (A) Chemical structure of CoA. (B) Schematic representation of the portions of the DR-CoAse structure that interact with CoA. (C) Surface representation of the binding site. Positive regions are in blue; negative regions are in red. (In panels B and C, helix α1 was omitted.).