Structure of Ddi2, a highly inducible detoxifying metalloenzyme from Saccharomyces cerevisiae

Abstract

Cyanamide (H₂N-CN) is used to break bud dormancy in woody plants and to deter alcohol use in humans. The biological effects of cyanamide in both these cases require the enzyme catalase. We previously demonstrated that Saccharomyces cerevisiae exposed to cyanamide resulted in strong induction of DDI2 gene expression. Ddi2 enzymatically hydrates cyanamide to urea and belongs to the family of HD-domain metalloenzymes (named after conserved active-site metal-binding His and Asp residues). Here, we report the X-ray structure of yeast Ddi2 to 2.6 Å resolution, revealing that Ddi2 is a dimeric zinc metalloenzyme. We also confirm that Ddi2 shares structural similarity with other known HD-domain proteins. HD residues His-55, His-88, and Asp-89 coordinate the active-site zinc, and the fourth zinc ligand is a water/hydroxide molecule. Other HD domain enzymes have a second aspartate metal ligand, but in Ddi2 this residue (Thr-157) does not interact with the zinc ion. Several Ddi2 active-site point mutations exhibited reduced catalytic activity. We kinetically and structurally characterized H137N and T157V mutants of Ddi2. A cyanamide soak of the Ddi2-T157V enzyme revealed cyanamide bound directly to the Zn²⁺ ion, having displaced the zinc-bound water molecule. The mode of cyanamide binding to Ddi2 resembles cyanamide binding to the active-site zinc of carbonic anhydrase, a known cyanamide hydratase. Finally, we observed that the sensitivity of ddi2Δ ddi3Δ to cyanamide was not rescued by plasmids harboring ddi2-H137N or ddi2-TI57V variants, demonstrating that yeast cells require a functioning cyanamide hydratase to overcome cyanamide-induced growth defects.

Keywords: HD domain; active site; crystallography; cyanamide; enzyme mechanism; enzyme structure; hydratase; metalloenzyme; urea; yeast physiology.

Figure 1.

Tertiary and quaternary structures of budding yeast Ddi2. A, ribbon diagram of the Ddi2 monomer with secondary structures labeled as in the multiple sequence alignment in Fig. 2. The zinc ion is shown as a gray sphere. Rainbow colors are shown for the secondary structure: the N terminus is blue, and the C terminus is red. Side chains for residues His-55, His-88, Asp-89, His-137, Gln-138, and Thr-157 near the zinc-binding site are depicted as stick models with nitrogen in blue and oxygen in red. B, drawn as for A but showing the observed dimer. The panels were drawn with PyMOL (42).

Figure 2.

Multiple sequence alignment of fungal orthologs of Ddi2. The sequence alignment was calculated using ClustalW (43), and the figure was prepared using ESPript 3.0 (44), based on the refined Ddi2 atomic coordinates. Secondary structures are depicted as follows: α represents α-helix, β represents β-sheet, and η represents 3¹⁰-helix. HD residues coordinating the zinc ion are indicated by black stars. The source organism abbreviations are given in the left-hand column. Sc, S. cerevisiae; Mv, M. verrucaria; Fg, Fusarium graminearum; An, A. nidulans; Ca, Candida albicans; Ao, A. oryzae; Dh, Debaryomyces hansenii. Identical residues are colored red. Conserved HD residues are highlighted by black stars, and residues chosen for site-specific mutagenesis are indicated by blue ovals. Residues involved in dimerization are highlighted by green triangles.

Figure 3.

Superposition of the Ddi2 and YpgQ HD-domain motifs. A, schematic ribbon depiction of Ddi2 chain D (conserved HD motif in blue, helix α-N in magenta, and other segments in green). The secondary structures are labeled as in Fig. 2. B, superposition of the HD motif of Ddi2 (blue) onto the HD motif of YpgQ (orange, PDB code 5DQV), highlighting the conserved five conserved α-helices: αA–αE (in this case a sixth N-terminal helix α-N (magenta) is also conserved). The zinc atom of Ddi2 is shown as a blue sphere, and the bound water is shown as a red sphere. C, as in B, but looking from the top at the Ddi2 active site. Conserved active-site residues (including Arg-58 and Asp-160) are shown as stick models colored by atom type for each model. Residue equivalences and root-mean-square deviation values for the superposition are given in Table S2 in the supplementary information. The panels were drawn with PyMOL (42).

Figure 4.

Active site of Ddi2 and cyanamide binding in the T157V mutant. A, NCS symmetry averaged |F_o| − |F_c| omit electron density map for chains A–G of the refined model (density for chains H and I is significantly weaker), using chain D as the reference subunit, with active-site solvent molecules omitted from the model (magenta, density displayed at 10.0 σ), showing the difference electron density near the active site (chain D). Conserved solvent molecules Wat-1–Wat-4 (red spheres) have been refined into the difference density. B, as in A, but showing the cyanamide (CYA) soak for the Ddi2-T157V mutant. The placed cyanamide substrate is depicted as a stick model. Side chains of residues are colored by element: oxygen in red, carbon in green, and nitrogen in blue. Bonds to the zinc ion are represented as yellow dashes. The zinc ion (cyan) and water molecules (red) are represented as spheres. The NCS maps were calculated using the NCS averaging option in Coot (40) and drawn with PyMOL (42).

Figure 5.

Identification and kinetic characterization of Ddi2-H137N and Ddi2-T157V mutants. A, cyanamide depletion assays with Ddi2-His₆ point mutations as labeled. Cyanamide depletion assay is described under “Experimental procedures.” B, enzyme kinetic analysis of Ddi2-T157V (black diamonds) and Ddi2-H137N (open circles) mutant proteins purified from GST-fusion proteins. Michaelis–Menten curves fitted to the data with nonlinear least squares regression as implemented in Prism are shown as solid black lines. The assays were carried out in duplicate, and standard errors for initial velocity measurements are indicated with black bars. For the kinetic assays, typically 1 μg of purified recombinant protein is used, but in the case of the H137N mutant, 100 μg was used to obtain measurable initial velocities.

Figure 6.

Analysis of cyanamide binding in the Ddi2 T157V mutant. A, superposition of chain D of WT Ddi2 (green) and Ddi2-T157V (cyan) showing active-site residues, solvent molecules, or bound cyanamide. B, view depicting the active site in chain D but viewing from the top right corner in A. The zinc ion is represented as a green (WT) or cyan (T157V) sphere, water molecules are represented as light red spheres, and cyanamide (CYA) is depicted as a stick model. The side chains of active-site residues are colored by element: oxygen is red, carbon is green, and nitrogen is blue. The panels were drawn with PyMOL (42).

Figure 7.

Rescue of cyanamide sensitivity of a ddi2 ddi3Δ mutant by WT Ddi2 or its active-site mutant derivatives. WT strain BY4741 and its ddi2Δ ddi3Δ mutant derivative WXY3149 were transformed with plasmids as indicated and used for a gradient plate assay as described under “Experimental procedures.” The plates were incubated for 48 h before taking the photograph. Only a single representative clone for each strain is shown on the plates. The arrow points in the direction of increasing cyanamide concentration.