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

Abstract

The Toblerone fold was discovered recently when the first structure of the cyclic amide hydrolase, AtzD (a cyanuric acid hydrolase), was elucidated. We surveyed the cyclic amide hydrolase family, finding a strong correlation between phylogenetic distribution and specificity for either cyanuric acid or barbituric acid. One of six classes (IV) could not be tested due to a lack of expression of the proteins from it, and another class (V) had neither cyanuric acid nor barbituric acid hydrolase activity. High-resolution X-ray structures were obtained for a class VI barbituric acid hydrolase (1.7 Å) from a Rhodococcus species and a class V cyclic amide hydrolase (2.4 Å) from a Frankia species for which we were unable to identify a substrate. Both structures were homologous with the tetrameric Toblerone fold enzyme AtzD, demonstrating a high degree of structural conservation within the cyclic amide hydrolase family. The barbituric acid hydrolase structure did not contain zinc, in contrast with early reports of zinc-dependent activity for this enzyme. Instead, each barbituric acid hydrolase monomer contained either Na ϩ or Mg 2ϩ , analogous to the structural metal found in cyanuric acid hydrolase. The Frankia cyclic amide hydrolase contained no metal but instead formed unusual, reversible, intermolecular vicinal disulfide bonds that contributed to the thermal stability of the protein. The active sites were largely conserved between the three enzymes, differing at six positions, which likely determine substrate specificity.IMPORTANCE The Toblerone fold enzymes catalyze an unusual ring-opening hydrolysis with cyclic amide substrates. A survey of these enzymes shows that there is a good correlation between physiological function and phylogenetic distribution within this family of enzymes and provide insights into the evolutionary relationships between the cyanuric acid and barbituric acid hydrolases. This family of enzymes is structurally and mechanistically distinct from other enzyme families; however, to date the structure of just two, physiologically identical, enzymes from this family has been described. We present two new structures: a barbituric acid hydrolase and an enzyme of unknown function. These structures confirm that members of the CyAH family have the unusual Toblerone fold, albeit with some significant differences.KEYWORDS atrazine, evolution, hydrolase, phylogenetic analysis, structure-activity relationships, triazine I n 2013, we described a unique fold for AtzD, the cyanuric acid hydrolase (CAH) from Pseudomonas sp. strain ADP, with the first X-ray structure of this enzyme (1). CAH belongs to the recently identified cyclic amide hydrolase (CyAH) family, members of

An unexpected vestigial protein complex reveals the evolutionary origins of an s-triazine catabolic enzyme

“…viciae 3841 (BiuH) (AM236084.1) and barbituric acid hydrolase from Nocardioides sp. JS614 coding for the protein ABL81019 (ABL81019.1) were expressed in E. coli as described elsewhere (12,19).…”

“…1B) is also comprised of three hydrolases: AtzD, AtzE and AtzF. Unlike the upper pathway, these three enzymes are serine hydrolases; AtzD is an unusual Ser-Lys hydrolase and is the archetype of a recently discovered protein fold (the Toblerone fold; (11,12)), while AtzE and AtzF are Ser-cisSer-Lys hydrolases (1,13). The lower pathway progresses via the AtzD-mediated ring opening of cyanuric acid to form an unstable product, 1-carboxybiuret, which is known to spontaneously decarboxylate to biuret in an aqueous environment (1).…”

“…Interestingly, these enzymatic products are unstable in an aqueous environment and can undergo spontaneous reactions; allophanate decarboxylates to urea (14), whilst carbamic acid decomposes to ammonia and carbon dioxide in water (13,15). AtzD and AtzF have been overproduced in heterologous expression systems, which has allowed them to be studied biochemically (14)(15)(16)(17) and structurally (11)(12)(13)18). However, expression and purification of AtzE in a heterologous system has been challenging (1) and structural and biochemical data had not been obtained previously.…”

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

“…In the last few years the first structures of a cyanuric acid hydrolase have been reported[8–10]. The two structures reported were for the enzymes from Pseudomonas sp.…”

Structural and biochemical characterization of the biuret hydrolase (BiuH) from the cyanuric acid catabolism pathway of Rhizobium leguminasorum bv. viciae 3841

“…AtzE and AtzF are Ser- cis Ser-Lys amidohydrolases and both possess an amidase-fold (PFAM PF01425); the structure of AtzF has been determined experimentally [ 15 , 19 ], while that of AtzE has been inferred from its sequence identity with other amidase proteins [ 14 ]. AtzD is also a serine hydrolase, but it belongs to a recently described structural family (the Toblerone fold; PFAM PF09663) that is unrelated to Ser- cis Ser-Lys hydrolases [ 10 , 11 , 20 ].…”