Figure 1.
CAH catalytic reaction and inhibitor structure.
A. CAH conversion of cyanuric acid to carboxybiuret, which spontaneously decarboxylates to produce biuret. B. Structure of the CAH substrate analog and inhibitor, barbituric acid.
Table 1.
Data collection and refinement statistics.
Figure 2.
A. The side view of monomer structure. The selenium SAD-phased electron density for the barbituric acid inhibitor is shown in blue mesh, contoured at 2.0 σ. The simulated-annealing omit Fo-Fc map with the barbituric acid inhibitor omitted is shown in magenta mesh, contoured at 3.0 σ. Domains are depicted in different colors: Domain A, consisting of residues 1–99, in blue; Domain B, consisting of residues 108–242, in green; and Domain C, consisting of residues 249–355 in hot pink. B. The top view of monomer structure compared to A, looking through the center of the barrel along the pseudo three-fold axis. C. Overlay of the three domains of CAH. D. Depiction of the trimeric repeat domain fold in CAH. Red cylinders represent α-helices and blue arrows represent β-strands. α-helices 2 and 3 are present in the domain B, while domains A and C have a loop in place of α2.
Figure 3.
Structural comparison between CAH and AtzD.
A. Comparison of overall structures between CAH and AtzD monomers. CAH is colored in green while AtzD is colored in magenta. B. Comparison of the active sites between CAH and AtzD showing highly similar arrangement of the residues involved in substrate binding and catalysis. Residue labels are for CAH.
Figure 4.
Comparison between CAH fold and the homotrimeric proteins in the YjgF-like SCOP structural superfamily.
A. The trimer of AroH-type chorismate mutase (CM) with active sites in each subunit interface that is accessed from the exterior of the barrel. B, C and D show the overlays of CAH domain A (residues 1–99, green) and a monomer of various structurally similar homotrimeric proteins (magenta): B. AroH-type CM (1COM). C. Proposed translation initiation inhibitor (PDB ID: 3GTZ). D. Putative aminoacrylate peracid reductase, RutC (PDB ID: 3V4D).
Figure 5.
Trimeric conglomeration of CAH active site.
A. Three serine residues (Ser79, Ser226, Ser333) lie within hydrogen bond distances from corresponding lysine residues (Lys 40, Lys 156, and Lys 285, respectively). Each pair shows the typical arrangement for the Ser/Lys dyad hydrolase. B. Three arginine residues (Arg 52, Arg 188, Arg 314) and the backbone nitrogens from Gly80, Ser227 and Gly334 form hydrogen bonds with the carbonyl oxygens of the barbituric acid inhibitor. The simulated annealing composite-omit 2Fo-Fc map is shown in colored mesh, contoured at1.5 σ.
Figure 6.
Sequence alignment showing conservation of three Ser/Lys dyads found within subgroups of the CAH/barbiturase family.
Subgroups of the updated family of 119 sequences were determined via phylogenetic clades. Consensus sequences were determined for each of the subgroups. Only sequences directly surrounding the dyads are displayed. Absolute, 100% conservation is represented by capital letters and greater than 50% conservation by lowercase letters. Columns in which no single residue was conserved at greater than 50% are depicted by a question mark. This usually occurred in regions of highly divergent sequence or where subgroups within a clade contained different residues. Residues equivalent to the Ser/Lys dyad residues are outlined with black boxes. Arrows point to Ser/Lys dyad residues with complete conservation across the family; residues not conserved in these positions are highlighted in red.
Table 2.
Specific activity of wild type and mutant CAH on cyanuric acid as determined by the biuret hydrolase-coupled ammonia detection assay*.
Figure 7.
Hydrogen bonding and electrostatic interactions network around three lysines from each domain in CAH.
A. Lys40 forms hydrogen bonds with the backbone carbonyls of Gly80 and Gly81, and the Ser79 side-chain. B. Lys156 is hydrogen-bonded to backbone carbonyls of Met78 and Ser227, and Ser226 side-chain. In addition, Glu230 forms electrostatic interaction with Lys156. C. Lys285 is hydrogen bonded to backbone carbonyls of Gly334 and Gly335, and Ser333 side-chain.
Figure 8.
After substrate binding, Ser226 attacks the substrate carbonyl, producing an tetrahedral intermediate. Arginine and a backbone amide group form an oxyanion hole to stabilize the negative charge on intermediates. The substrate carbonyl reforms with the cleavage of the C-N ring bond. Water then attacks the acyl intermediate to release the product.
Figure 9.
Front and back channels around the active site of CAH.
A. A view looking along the front channel down to the active site-bound barbituric acid. A second barbituric acid molecule, found on the surface is also shown in sticks with a simulated annealing composite omit 2Fo-Fc map contoured at 2.0 σ. B. Cut-away view of the CAH monomer. Barbituric acid is seen at the base of the front-side channel (red arrow). The back channel (blue arrow) is blocked by two hydrogen-bonds formed by Lys156 and Met78 (shown in sticks), and by Lys156 and Ser226, and therefore does not reach the substrate-binding site. C. Close-up view of the hydrogen bonds between Lys and Met78. D. Barbituric acid molecule bound near the entry of the front side channel, on the tetramer interface cleft.