Crystal structures of Moorella thermoacetica cyanuric acid hydrolase reveal conformational flexibility and asymmetry important for catalysis

Abstract

An ancient enzyme family responsible for the catabolism of the prebiotic chemical cyanuric acid (1,3,5-triazine-2,4,6-triol) was recently discovered and is undergoing proliferation in the modern world due to industrial synthesis and dissemination of 1,3,5-triazine compounds. Cyanuric acid has a highly stabilized ring system such that bacteria require a unique enzyme with a novel fold and subtle active site construction to open the ring. Each cyanuric acid hydrolase monomer consists of three isostructural domains that coordinate and activate the three-fold symmetric substrate cyanuric acid for ring opening. We have now solved a series of X-ray structures of an engineered, thermostable cyanuric acid ring-opening enzyme at 1.51 ~ 2.25 Å resolution, including various complexes with the substrate, a tight-binding inhibitor, or an analog of the reaction intermediate. These structures reveal asymmetric interactions between the enzyme and bound ligands, a metal ion binding coupled to conformational changes and substrate binding important for enzyme stability, and distinct roles of the isostructural domains of the enzyme. The multiple conformations of the enzyme observed across a series of structures and corroborating biochemical data suggest importance of the structural dynamics in facilitating the substrate entry and the ring-opening reaction, catalyzed by a conserved Ser-Lys dyad.

A procedure for removal of cyanuric acid in swimming pools using a cell-free thermostable cyanuric acid hydrolase

“…The six proteins included three represented in Fig. 1 , M. thermoacetica (WT and C46A) (accession ID: WP_011393610.1) (Shi et al., 2019 ), B. diazoefficiens (formerly Bradyrhizobium japonicum USDA110, accession ID: WP_011090016.1) (Seffernick et al., 2012 ) , Acidovorax citrulli (accession ID: WP_031302833.1) (Bera et al., 2017 ), and two putative CAH enzymes from Pseudolabrys sp. Root1462 (accession ID: WP_056911810.1) and Bradyrhizobium sp.…”

“…Structure superposition of CAH-PR and CAH-BD homology models with Pseudomonas sp. ADP (PDB:4BVQ) (Peat et al., 2013 ), M. thermoacetica CAH (PDB: 6BUM) (Shi et al., 2019 ), and A. citrulli (122227) TrzD (PDB: 5T13) (Bera et al., 2017 ) by molecular operating environment (Chemical Computing Group ULC, Montreal, QC, Canada) showed that these enzymes had very similar folding and overall structures (Fig. S4, generated by PyMOL [Yuan et al., 2016 ]).…”

“…S4, generated by PyMOL [Yuan et al., 2016 ]). The catalytic residues of CAH are typically defined by three groups of arginine–lysine–serine triads from three domains (Shi et al., 2019 ). Sequence alignment and structure superposition of CAH-PR and M. thermoacetica (6BUM) showed that these catalytic residues and the consensus sequence at the C-terminus were identical and at the same position, indicating that the observed thermostability was not due to structural variation within the active sites or the consensus sequence.…”

“…CAHs are members of a relatively small protein family that also includes barbiturases and several proteins of yet unknown function (Seffernick et al., 2012 ). They all share a unique fold consisting of three domains that circumscribe the active site (Cho et al., 2014 ; Peat et al., 2013 ; Shi et al., 2019 ). The active site is defined by each domain contributing a triad of arginine, lysine, and serine, which together assemble a threefold symmetrical binding site for the threefold symmetric CYA substrate.…”

Cyanuric Acid Biodegradation via Biuret: Physiology, Taxonomy, and Geospatial Distribution

“…Extensive biochemical characterization has revealed that the cyanuric acid hydrolases and barbiturases exhibit remarkably stringent substrate specificities, being reactive only with their specific substrates (14)(15)(16)(17)(18). The X-ray crystal structures of four cyanuric acid hydrolases and a barbiturase identified a unique protein fold with three protein domains defining a fairly symmetric active site (19)(20)(21)(22). The cyanuric acid hydrolase active site is composed of three symmetric Ser-Lys-Arg triads that define the singular substrate specificity (20).…”