Research Article: Cyanuric Acid Hydrolase from Azorhizobium caulinodans ORS 571: Crystal Structure and Insights into a New Class of Ser-Lys Dyad Proteins

Date Published: June 10, 2014

Publisher: Public Library of Science

Author(s): Seunghee Cho, Ke Shi, Jennifer L. Seffernick, Anthony G. Dodge, Lawrence P. Wackett, Hideki Aihara, Inês A. Cardoso Pereira.


Cyanuric acid hydrolase (CAH) catalyzes the hydrolytic ring-opening of cyanuric acid (2,4,6-trihydroxy-1,3,5-triazine), an intermediate in s-triazine bacterial degradation and a by-product from disinfection with trichloroisocyanuric acid. In the present study, an X-ray crystal structure of the CAH-barbituric acid inhibitor complex from Azorhizobium caulinodans ORS 571 has been determined at 2.7 Å resolution. The CAH protein fold consists of three structurally homologous domains forming a β-barrel-like structure with external α-helices that result in a three-fold symmetry, a dominant feature of the structure and active site that mirrors the three-fold symmetrical shape of the substrate cyanuric acid. The active site structure of CAH is similar to that of the recently determined AtzD with three pairs of active site Ser-Lys dyads. In order to determine the role of each Ser-Lys dyad in catalysis, a mutational study using a highly sensitive, enzyme-coupled assay was conducted. The 109-fold loss of activity by the S226A mutant was at least ten times lower than that of the S79A and S333A mutants. In addition, bioinformatics analysis revealed the Ser226/Lys156 dyad as the only absolutely conserved dyad in the CAH/barbiturase family. These data suggest that Lys156 activates the Ser226 nucleophile which can then attack the substrate carbonyl. Our combination of structural, mutational, and bioinformatics analyses differentiates this study and provides experimental data for mechanistic insights into this unique protein family.

Partial Text

Cyanuric acid, or 2,4,6-trihydroxy-1,3,5-triazine, is an industrially important compound used to make pesticides, dyes, and disinfectants. The latter consist largely of N-chlorinated cyanuric acid derivatives, used for cleaning and swimming pool disinfection. In 2005, 350 million pounds of cyanuric acid were produced industrially for those purposes [1]. Although cyanuric acid alone is not very toxic, coingestion with melamine was the cause of recent poisonings due to adulterated pet food [2], leading to the largest pet food recall in North America and a $24 million settlement.

The reported crystal structure of CAH has internal three-fold symmetry, originating from the concatenation of three homologous domains. Each domain contributes equivalent active site residues, producing a three-fold symmetry within the active site. The symmetry of the cyanuric acid substrate extends this theme to the active site’s substrate binding. Geometry and substrate analog positioning within the active site indicates that CAH likely utilizes a Ser-Lys dyad for catalysis. However, as there are three Ser-Lys dyads in the active site, additional analyses were required to address their specific roles.