Sequence alignment and the overall conformation of AllD in the monomeric and dimeric structure.

<p><b>A</b>, The amino acid sequences of AllD are compared with members of the NAD(P)H-dependent oxidoreductase family with known structures: TMLDH annotated as <i>Thermus thermophilus</i> HB8 Type 2 malate/lactate dehydrogenase (1VBI; Z-score, 45.3; rmsd, 1.5 Å), AMDH annotated as <i>Agrobacterium tumefaciens</i> malate dehydrogenase (1Z2I; Z-score, 44.3; rmsd, 1.5 Å), SLDH for <i>Methanocaldococcus </i>l-sulfolactate dehydrogenase (2X06; Z-score, 41.6; rmsd, 1.9 Å) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052066#pone.0052066-Irima1" target="_blank">[23]</a>, PMDH annotated as <i>Pyrococcus horikoshii</i> OT3 malate dehydrogenase (1V9N; Z-score, 41.1; rmsd, 1.6 Å), EMDH annotated as <i>Entamoeba histolytica</i> malate dehydrogenase (3I0P; Z-score, 40.6; rmsd, 2.1 Å), DpkA for <i>Pseudomonas syringae</i> Δ¹-piperideine-2-carboxylate/Δ¹-pyrroline-2-carboxylate reductase (2CWF; Z-score, 37.4; rmsd, 2.2 Å) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052066#pone.0052066-Goto1" target="_blank">[24]</a>, EMLDH annotated as <i>E. coli</i> malate/l-lactate dehydrogenases (2G8Y; Z-score, 37.0; rmsd, 2.4 Å), YiaK for <i>E. coli</i> 2,3-diketo-l-gulonate reductase (1S20; Z-score, 36.1; rmsd, 2.7 Å) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052066#pone.0052066-Forouhar1" target="_blank">[25]</a>. 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052066#pone.0052066-Gouet1" target="_blank">[31]</a>. <b>B</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. <b>C</b>, A dimer in the asymmetric unit of the binary complex with NADH is displayed, with helices in the intersubunit interface.</p

Binding site for glyoxylate and NADH-induced conformational changes.

<p><b>A</b>, Glyoxylate, with a <i>Fo-Fc</i> 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>B</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. <b>C</b>, Conformational changes are observed in Domain III of the binary (red) and ternary (blue) complex compared to that in the apo form (black).</p

Structural and Functional Insights into (<em>S</em>)-Ureidoglycolate Dehydrogenase, a Metabolic Branch Point Enzyme in Nitrogen Utilization

<div><p>Nitrogen metabolism is one of essential processes in living organisms. The catabolic pathways of nitrogenous compounds play a pivotal role in the storage and recovery of nitrogen. In <em>Escherichia coli</em>, two different, interconnecting metabolic routes drive nitrogen utilization through purine degradation metabolites. The enzyme (<em>S</em>)-ureidoglycolate dehydrogenase (AllD), which is a member of l-sulfolactate dehydrogenase-like family, converts (<em>S</em>)-ureidoglycolate, a key intermediate in the purine degradation pathway, to oxalurate in an NAD(P)-dependent manner. Therefore, AllD is a metabolic branch-point enzyme for nitrogen metabolism in <em>E. coli</em>. Here, we report crystal structures of AllD in its apo form, in a binary complex with NADH cofactor, and in a ternary complex with NADH and glyoxylate, a possible spontaneous degradation product of oxalurate. Structural analyses revealed that NADH in an extended conformation is bound to an NADH-binding fold with three distinct domains that differ from those of the canonical NADH-binding fold. We also characterized ligand-induced structural changes, as well as the binding mode of glyoxylate, in the active site near the NADH nicotinamide ring. Based on structural and kinetic analyses, we concluded that AllD selectively utilizes NAD⁺ as a cofactor, and further propose that His116 acts as a general catalytic base and that a hydride transfer is possible on the B-face of the nicotinamide ring of the cofactor. Other residues conserved in the active sites of this novel l-sulfolactate dehydrogenase-like family also play essential roles in catalysis.</p> </div

Scheme for conversion of (<i>S</i>)-ureidoglycolate via three different enzymes.

<p>Scheme for conversion of (<i>S</i>)-ureidoglycolate via three different enzymes.</p

Enzyme assay of the wild-type AllD and its mutants.

a<p>Values in parentheses are standard error.</p>b<p>Activity was not detected even at 1.5 µM (50 µg/mL) of enzyme.</p

Interactions between NADH and AllD.

<p><b>A</b>, NADH is located at the inter-domain interface, with the <i>Fo-Fc</i> electron density map contoured at 3σ. Residues for the NADH-binding site are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052066#pone-0052066-g002" target="_blank">Figure 2A</a> and displayed by the color codes used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052066#pone-0052066-g002" target="_blank">Figure 2B</a>, except for residues in gray, which represent interactions between other subunits. <b>B</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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052066#pone-0052066-g002" target="_blank">Figure 2A</a>).</p

Data collection and refinement statistics.

a<p>High-resolution cutoff was based on the CC_1/2 value obtained from the program Aimless in CCP4 suite <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052066#pone.0052066-Karplus1" target="_blank">[32]</a>.</p>b<p>Numbers in parentheses refer to data in the highest resolution shell.</p>c<p><i>R_merge =</i>Σ|I_h−h>|/Σ I_h, where I<i>_h</i> is the observed intensity and _h> is the average intensity.</p>d<p><i>R_work</i> = Σ ||F_obs|-k|F_cal||/Σ|F_obs|.</p>e<p><i>R_free</i> is the same as <i>R_obs</i> for a selected subset (10%) of the reflections that was not included in prior refinement calculations.</p>f<p>Ordered residues: apo structure (Met1 to Tyr337 in subunit A and Ile3 to Ala317 in subunit B), a binary complex (Met1 to Tyr337 in subunit A and Ile3 to Lys315 in subunit B), and a ternary complex (Met1 to Asn338 in subunit A and Ser4 to Lys315 in subunit B).</p>g<p>Outliers identified using a program MolProbity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052066#pone.0052066-Chen1" target="_blank">[33]</a>: three residues in apo form, Ser333 for subunit A, Asn157, Asn192 for subunit B; two residues in the binary complex, Asn157 for subunit A, Asn157 for subunit B; four residues in the ternary complex, Arg113, Asn157 for subunit A, Met143, Asn157 for subunit B.</p

Gene Context Analysis Reveals Functional Divergence between Hypothetically Equivalent Enzymes of the Purine–Ureide Pathway

A major problem of genome annotation is the assignment of a function to a large number of genes of known sequences through comparison with a relatively small number of experimentally characterized genes. Because functional divergence is a widespread phenomenon in gene evolution, the transfer of a function to homologous genes is not a trivial exercise. Here, we show that a family of homologous genes which are found in purine catabolism clusters and have hypothetically equivalent functions can be divided into two distinct groups based on the genomic distribution of functionally related genes. One group (UGLYAH) encodes proteins that are able to release ammonia from (<i>S</i>)-ureidoglycine, the enzymatic product of allantoate amidohydrolase (AAH), but are unable to degrade allantoate. The presence of a gene encoding UGLYAH implies the presence of AAH in the same genome. The other group (UGLYAH2) encodes proteins that are able to release ammonia from (<i>S</i>)-ureidoglycine as well as urea from allantoate. The presence of a gene encoding UGLYAH2 implies the absence of AAH in the same genome. Because (<i>S</i>)-ureidoglycine is an unstable compound that is only formed by the AAH reaction, the <i>in vivo</i> function of this group of enzymes must be the release of urea from allantoate (allantoicase activity), while ammonia release from (<i>S</i>)-ureidoglycine is an accessory activity that evolved as a specialized function in a group of genes in which the coexistence with AAH was established. Insights on the active site modifications leading to a change in the enzyme activity were provided by comparison of three-dimensional structures of proteins belonging to the two different groups and by site-directed mutagenesis. Our results indicate that when the neighborhood of uncharacterized genes suggests a role in the same process or pathway of a characterized homologue, a detailed analysis of the gene context is required for the transfer of functional annotations

Restoration of root growth inhibition phenotype by complementation.

<p>Col-0, <i>coi1-1</i> heterozygote, and complemented T3 homozygous lines were grown vertically on MS media containing 50 µM MeJA (left) or without MeJA (right). The asterisk indicates <i>coi1-1</i> homozygote which was tested by PCR and <i>Xcm</i>I enzyme digestion.</p

Morphological phenotype of complemented <i>coi1-1</i>.

<p>The <i>coi1-1</i> mutant was complemented with <i>OsCOI1a</i> (#1134), <i>OsCOI1b</i> (#2124), <i>OsCOI2</i>(H391Y) (#H3159) or <i>COI1</i> (#A0088), respectively, at the T2 generation. A, Siliques of 6-week-old plants grown in soil. The asterisks indicate developing siliques. B, Flowers of 6-week-old plants grown in soil. C, Fully developed siliques. D, Developing seeds in the silique.</p