Fig 1.
Cyanuric acid mineralization by bacteria.
Cyanuric acid is mineralized to CO2 and NH3 by cyanuric acid mineralizing bacteria by the enzymes cyanuric acid amidohydrolase, biuret hydrolase and allophanate hydrolase. The product of cyanuric acid amiidohydrolase (1-carboxybiuret) is unstable under physiological conditions and decarboxylates to form biuret and CO2. The product of allophanate amidohydrolase (dicarboxyammonia) is also unstable under physiological conditions and decomposes to CO2 and NH3.
Table 1.
Data collection and refinement statistics.
Fig 2.
Structure of the biuret amidohydrolase enzyme.
Cartoon representation of: A. BiuH tetrameric structure, each color represents a subunit; B. monomeric subunit of BiuH with alpha helices shown in red (numbered α1- α8), beta strands in yellow (numbered β1- β5) and loops in green. In every cases the N and/or C termini were visible, they are indicated by a black arrow. Figures 1, 3 and 6 were generated with PyMol [65].
Fig 3.
Reactions catalyzed by biuret amidohydrolase and homologs involved in heterocycle catabolism.
1) Ureidoacrylate peracid amidohydrolase (RutB) produces carbamate and peroxy-amino acrylate from peroxy-ureidoacrylate, which is produced by ring opening of uracil by RutA and RutF; 2) maleamate amidohydrolase (NicF) produces ammonia and maleic acid from maleamaic acid, produced by NicX and NicD from 2,5-dihydroxypyridine during nicotinic acid catabolism; and, 3) biuret amidohydrolase (BiuH) produces ammonia and allophanate from biuret during cyanuric acid catabolism.
Fig 4.
Cys175Ser BiuH variant showing biuret in the active site.
BiuH is represented in cartoon style, with the exception of the active site amino acids; biuret is shown in pink, the hydrogen bonds between the residues and biuret are shown in grey, the hydrogen bonds between Asp36 and Lys142 in red; Gln215 is shown in cyan as it belongs to another enzyme subunit. The difference density map (Fo −Fc) of biuret in the active site is shown in S11 Fig.
Fig 5.
Suggested mechanism of the BiuH.
Lys142 stabilizes Asp36 that will act as a general base and deprotonate Cys175, allowing Cys175 to perform a nucleophilic attack on the carbonyl end of biuret. Cys175 then binds to biuret forming a tetrahedral intermediate. Asp36 then acts as a general acid, leading to the collapse of the intermediate and the production of an ammonia and a thioester intermediate. Following the addition of a water molecule, Asp36 deprotonates the molecule of water leading to the hydrolysis of the thioester intermediate, forming a new tetrahedral intermediate. Finally, the enzyme is restored to its original state, releasing the allophanate product.
Fig 6.
Specific activity of variant enzymes compared to the BiuH wild type enzyme.
The specific activity is shown for each variant as percentage of the wild type BiuH specific activity, using 1.2 mM biuret as substrate (n = 3).
Fig 7.
The active site of the Lys142Ala variant of BiuH showing Cys175 bound to the inhibitor N-carbamoyl-D,L-aspartic acid.
BiuH is represented in cartoon style, with the exception of the active site amino acids; N-carbamoyl-D,L-aspartic acid is shown in pink, the hydrogen bond between the Gln215 and N-carbamoyl-D,L-aspartic acid are shown in grey, the hydrogen bonds between Asp36 and Ala142 in red; Gln215 is shown in cyan as it belongs to another enzyme subunit. The inhibition profile of BiuH with N-carbamoyl-D,L-aspartic acid is shown in S5 Fig and the difference density map (Fo −Fc) of the covalently bound inhibitor is shown in S11 Fig.
Table 2.
Steady state kinetic parameters of BiuH and its variants.
(n = 5).
Fig 8.
Solvent accessible channels in BiuH.
Three solvent accessible channels emerging from the active site are shown in blue, green, and yellow. A) The blue and green tunnels quickly reach the surface of the monomer, while the yellow tunnel extends into the subunit interface and central cavity of the tetramer. The amino acids constituting the active site are shown as sticks, and the subunit that contained biuret is shown in pale green. B) The active site residues are shown as green sticks (for a representative structure of cluster 0) and as grey lines (for representative structures of other 19 clusters) and biuret is shown as Van der Waals spheres. The residues that are responsible for gating these tunnels are labeled in red C) The three dominant tunnels are shown on the right (green, yellow, and blue). Figure generated with Caver [52].