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

Abstract

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.

Bacterial Cyanuric Acid Hydrolase for Water Treatment

“…These results are consistent with in vitro studies comparing CAH to AtzD and TrzD, in which CAH was shown to be more thermally stable (16). Circular dichroism (CD) spectroscopy has shown that the thermal denaturation temperature of purified CAH in buffer is above 70°C (10).…”

“…Of these, cyanuric acid hydrolase is the most well studied and has been found to be, along with the enzyme barbiturase, a member of a rare protein family that is distinct from major hydrolase superfamilies (8). The X-ray structures of two cyanuric acid hydrolases were recently determined and were found to have a protein fold unique to the cyanuric acid/barbiturase protein family (10,11). Characterized cyanuric acid hydrolases were all from bacteria until a recent study described a homologous, isofunctional enzyme from a fungus (12).…”

High-Resolution X-Ray Structures of Two Functionally Distinct Members of the Cyclic Amide Hydrolase Family of Toblerone Fold Enzymes

“…To date, the only crystal structures from the CyAH family have been the CAHs from Pseudomonas sp. strain ADP and A. caulinodans (1,2), which have a high degree of sequence and structural similarity to each other. In the work presented here, we have empirically determined the structures of a BAH and the CyAH from Frankia ( Fig.…”

“…It is noteworthy that this Glu297/301 is just two residues removed from the conserved lysine in the third Ser-Lys pair of the active site, and we previously suggested that the structural metal of CAH was important in stabilizing this component of the active site (1). The conserved metal binding residues are found in both the Pseudomonas CAH as well as in the 4NQ3 structure of CAH from A. caulinodans (2). The Frankia CyAH also lacks the metal-coordinating residues Ala347 and Gln349.…”

“…Crystallographic and proteomic evidence suggested that Ser85 and Lys42 were the catalytic dyad; however, genetic and phylogenetic evidence presented in a later study suggest that Ser226 and Lys156 comprise the catalytic center (1,2).…”