Abstract
Cyanuric acid hydrolases are of industrial importance because of their use in aquatic recreational facilities to remove cyanuric acid, a stabilizer for the chlorine. Degradation of excess cyanuric acid is necessary to maintain chlorine disinfection in the waters. Cyanuric acid hydrolase opens the cyanuric acid ring hydrolytically and subsequent decarboxylation produces carbon dioxide and biuret. In the present study, we report the X-ray structure of TrzD, a cyanuric acid hydrolase from Acidovorax citrulli. The crystal structure at 2.19 Å resolution shows a large displacement of the catalytic lysine (Lys163) in domain 2 away from the active site core, whereas the two other active site lysines from the two other domains are not able to move. The lysine displacement is proposed here to open up a channel for product release. Consistent with that, the structure also showed two molecules of the co-product, carbon dioxide, one in the active site and another trapped in the proposed exit channel. Previous data indicated that the domain 2 lysine residue plays a role in activating an adjacent serine residue carrying out nucleophilic attack, opening the cyanuric acid ring, and the mobile lysine guides products through the exit channel.
Highlights
Cyanuric acid hydrolases are rare, ancient enzymes undergoing a modern renaissance due to the annual production of more than one billion pounds of s-triazine ring compounds that are microbially biodegraded via cyanuric acid as a metabolic intermediate1. s-Triazine rings are not known to be biosynthesized, but cyanuric acid forms from abiotic chemistry[2], and their facile synthesis from cheap precursors has spurred industrial production of more than one hundred s-triazine compounds
The enzyme is proposed to catalyze an opening of the s-triazine ring via a nucleophilic serine residue that is activated by a nearby lysine, a mechanistic motif known as a serine-lysine dyad[7,8]
Cyanuric acid hydrolases are a unique class of proteins structurally, showing a pseudo three-fold symmetry
Summary
Cyanuric acid hydrolases are rare, ancient enzymes undergoing a modern renaissance due to the annual production of more than one billion pounds of s-triazine ring compounds that are microbially biodegraded via cyanuric acid as a metabolic intermediate1. s-Triazine rings are not known to be biosynthesized, but cyanuric acid forms from abiotic chemistry[2], and their facile synthesis from cheap precursors has spurred industrial production of more than one hundred s-triazine compounds. One group has proposed a serine residue in domain one as the most likely nucleophile[10], and the other group has suggested the serine in domain two to be the putative nucleophile[9] This different interpretation remains unresolved to date because the three active site serine-lysine dyads have such a high degree of symmetry that discerning differential functions has yet not been possible. In the course of the study, we observed an unusual orientation of the second-domain lysine that interacts with the proposed catalytic second-domain serine This observation simultaneously provided support that the second domain serine serves as the active site nucleophile and it gave evidence for a product exit channel. The presence of trapped carbon dioxide in the enzyme is consistent with the view that the enzyme assists in the decarboxylation of carboxybiuret, and the cell does not rely on a spontaneous decarboxylation to produce the intermediate, biuret, in the cyanuric acid biodegradation pathway (Fig. 1)
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