Figure 1.
Scheme for conversion of (S)-ureidoglycolate via three different enzymes.
Table 1.
Data collection and refinement statistics.
Figure 2.
Sequence alignment and the overall conformation of AllD in the monomeric and dimeric structure.
A, The amino acid sequences of AllD are compared with members of the NAD(P)H-dependent oxidoreductase family with known structures: TMLDH annotated as Thermus thermophilus HB8 Type 2 malate/lactate dehydrogenase (1VBI; Z-score, 45.3; rmsd, 1.5 Å), AMDH annotated as Agrobacterium tumefaciens malate dehydrogenase (1Z2I; Z-score, 44.3; rmsd, 1.5 Å), SLDH for Methanocaldococcus l-sulfolactate dehydrogenase (2X06; Z-score, 41.6; rmsd, 1.9 Å) [23], PMDH annotated as Pyrococcus horikoshii OT3 malate dehydrogenase (1V9N; Z-score, 41.1; rmsd, 1.6 Å), EMDH annotated as Entamoeba histolytica malate dehydrogenase (3I0P; Z-score, 40.6; rmsd, 2.1 Å), DpkA for Pseudomonas syringae Δ1-piperideine-2-carboxylate/Δ1-pyrroline-2-carboxylate reductase (2CWF; Z-score, 37.4; rmsd, 2.2 Å) [24], EMLDH annotated as E. coli malate/l-lactate dehydrogenases (2G8Y; Z-score, 37.0; rmsd, 2.4 Å), YiaK for E. coli 2,3-diketo-l-gulonate reductase (1S20; Z-score, 36.1; rmsd, 2.7 Å) [25]. Highly conserved residues are shown in red and boxed in blue; strictly conserved residues are shown on a red background. Red triangles represent the residues involved in binding of NADH at the active site, while residues for the glyoxylate-binding site are indicated by blue asterisks. The secondary structural elements defined in an apo form are shown for the corresponding AllD sequences, with Domains I, II, III in cyan, orange, and magenta, respectively. These color codes are used throughout the manuscript, and the figure was prepared using ESPript [31]. B, The overall structure of monomeric AllD is shown, displaying the secondary structure elements with each domain in different colors. The molecule was orientated so that the inter-domain interface is located at the center of the monomer. C, A dimer in the asymmetric unit of the binary complex with NADH is displayed, with helices in the intersubunit interface.
Figure 3.
Interactions between NADH and AllD.
A, NADH is located at the inter-domain interface, with the Fo-Fc electron density map contoured at 3σ. Residues for the NADH-binding site are indicated in Figure 2A and displayed by the color codes used in Figure 2B, except for residues in gray, which represent interactions between other subunits. B, Schematic diagram showing the NADH-binding mode in the active site. The dashed lines indicate putative hydrogen bonds, which are labeled with the interatomic distance in Å; other residues represent van der Waals interactions of less than 5.0 Å. Water molecules are shown as red spheres. Residues are indicated by color coding, and underlined if they are conserved within the family (see Figure 2A).
Figure 4.
Binding site for glyoxylate and NADH-induced conformational changes.
A, Glyoxylate, with a Fo-Fc electron density map contoured at 1σ, is shown with the nearby residues within a distance of 5.0 Å. NADH is indicated in magenta. Ser43 and Tyr52 form hydrogen bonds with His44 and His116, respectively. B, Schematic view for the binding site of glyoxylate. In this scheme, glyoxylate-interacting residues in the first and second shell are shown, along with the possible hydrogen-bonding network in those residues. Water molecules are indicated by the red circle, as seen in a. C, Conformational changes are observed in Domain III of the binary (red) and ternary (blue) complex compared to that in the apo form (black).
Table 2.
Enzyme assay of the wild-type AllD and its mutants.