The cyanuric acid hydrolase, AtzD, is the founding member of a newly identified family of ring-opening amidases. We report the first X-ray structure for this family, which is a novel fold (termed the ‘Toblerone’ fold) that likely evolved via the concatenation of monomers of the trimeric YjgF superfamily and the acquisition of a metal binding site. Structures of AtzD with bound substrate (cyanuric acid) and inhibitors (phosphate, barbituric acid and melamine), along with mutagenesis studies, allowed the identification of the active site. The AtzD monomer, active site and substrate all possess threefold rotational symmetry, to the extent that the active site possesses three potential Ser–Lys catalytic dyads. A single catalytic dyad (Ser85–Lys42) is hypothesized, based on biochemical evidence and crystallographic data. A plausible catalytic mechanism based on these observations is also presented. A comparison with a homology model of the related barbiturase, Bar, was used to infer the active-site residues responsible for substrate specificity, and the phylogeny of the 68 AtzD-like enzymes in the database were analysed in light of this structure–function relationship.

Pinoresinol is a dimer of two β-β'-linked coniferyl alcohol molecules. It is both a plant defense molecule synthesized through the shikimic acid pathway and a representative of several β-β-linked dimers produced during the microbial degradation of lignin in dead plant material. Until now, little has been known about the bacterial catabolism of such dimers. Here we report the isolation of the efficient (+)-pinoresinol-mineralizing sp. strain SG-MS2 and its catabolic pathway. Degradation of pinoresinol in this strain is inducible and proceeds via a novel oxidative route, which is in contrast to the previously reported reductive transformation by other bacteria. Based on enzyme assays and bacterial growth, cell suspension, and resting cell studies, we provide conclusive evidence that pinoresinol degradation in strain SG-MS2 is initiated by benzylic hydroxylation, generating a hemiketal via a quinone methide intermediate, which is then hydrated at the benzylic carbon by water. The hemiketal, which stays in equilibrium with the corresponding keto alcohol, undergoes an aryl-alkyl cleavage to generate a lactone and 2-methoxyhydroquinone. While the fate of 2-methoxyhydroquinone is not investigated further, it is assumed to be assimilated by ring cleavage. The lactone is further metabolized via two routes, namely, lactone ring cleavage and benzylic hydroxylation via a quinone methide intermediate, as described above. The resulting hemiketal again exists in equilibrium with a keto alcohol. Our evidence suggests that both routes of lactone metabolism lead to vanillin and vanillic acid, which we show can then be mineralized by strain SG-MS2. The oxidative catabolism of (+)-pinoresinol degradation elucidated here is fundamentally different from the reductive cometabolism reported for two previously characterized bacteria. Our findings open up new opportunities to use lignin for the biosynthesis of vanillin, a key flavoring agent in foods, beverages, and pharmaceuticals, as well as various new lactones. Our work also has implications for the study of new pinoresinol metabolites in human health. The enterodiol and enterolactone produced through reductive transformation of pinoresinol by gut microbes have already been associated with decreased risks of cancer and cardiovascular diseases. The metabolites from oxidative metabolism we find here also deserve attention in this respect.

dThe activity of the allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, provides the final hydrolytic step for the mineralization of s-triazines, such as atrazine and cyanuric acid. Indeed, the action of AtzF provides metabolic access to two of the three nitrogens in each triazine ring. The X-ray structure of the N-terminal amidase domain of AtzF reveals that it is highly homologous to allophanate hydrolases involved in a different catabolic process in other organisms (i.e., the mineralization of urea). The smaller C-terminal domain does not appear to have a physiologically relevant catalytic function, as reported for the allophanate hydrolase of Kluyveromyces lactis, when purified enzyme was tested in vitro. However, the C-terminal domain does have a function in coordinating the quaternary structure of AtzF. Interestingly, we also show that AtzF forms a large, ca. 660-kDa, multienzyme complex with AtzD and AtzE that is capable of mineralizing cyanuric acid. The function of this complex may be to channel substrates from one active site to the next, effectively protecting unstable metabolites, such as allophanate, from solvent-mediated decarboxylation to a dead-end metabolic product.A trazine (1-chloro-3-ethylamino-5-isopropylamino-2,4,6-triazine; Fig. 1) is one of the most heavily applied herbicides in the world and is registered for use in North and South America, Australia, Africa, Asia, and the Middle East. Atrazine is environmentally persistent (half-life, 4 to 57 weeks, depending on the location) and mobile, leading to the detection of atrazine in surface water, groundwater, and aquifers (1-3). Atrazine has been detected in the environment at concentrations of up to 4.6 M in several countries (2, 3). It has been suggested that atrazine may be a carcinogen and an endocrine disrupter at such concentrations (4-6).Since atrazine was introduced in the 1950s, bacteria have evolved highly efficient catabolic pathways that allow the use of atrazine as a sole nitrogen and carbon source (7-10). These pathways have provided valuable insights into the evolutionary processes that drive the establishment of new enzyme function and new catabolic pathways (11-15). In addition, these pathways and cognate enzymes provide a potential biotechnological solution to atrazine contamination (i.e., bioremediation) (16-19).The most intensively studied atrazine catabolism pathway was discovered in Pseudomonas sp. strain ADP in the mid-1990s and is comprised of six hydrolases: atrazine chlorohydrolase (AtzA; EC 3.8.1.8) (20,21), N-ethylaminohydrolase (AtzB; EC 3.5.99.3) (22,23), N-isopropylammelide isopropylaminohydrolase (AtzC; EC 3.5.99.4) (24, 25), cyanuric acid amidohydrolase (AtzD; EC 3.5.2.15) (15,26,27), biuret amidohydrolase (AtzE; EC 3.5.1.84) (28), and allophanate hydrolase (AtzF; EC 3.5.1.54) (29-31). These hydrolases sequentially dechlorinate (AtzA) and remove the two N-alkyl side groups (AtzB and AtzC) to produce cyanuric acid, which is then further hydrolyzed to biuret, allophanate, and ammonia via AtzD, AtzE, a...