New Family of Biuret Hydrolases Involved in s-Triazine Ring Metabolism

Abstract

Biuret is an intermediate in the bacterial metabolism of s-triazine ring compounds and is occasionally used as a ruminant feed supplement. We used bioinformatics to identify a biuret hydrolase, an enzyme that has previously resisted efforts to stabilize, purify and characterize. This newly discovered enzyme is a member of the cysteine hydrolase superfamily, a family of enzymes previously not found to be involved in s-triazine metabolism. The gene from Rhizobium leguminosarum bv. viciae strain 3841 encoding biuret hydrolase was synthesized, transformed into Escherichia coli, and expressed. The enzyme was purified and found to be stable. Biuret hydrolase catalyzed the hydrolysis of biuret to allophanate and ammonia. The kcat/KM of 1.7 × 105 M−1s−1 and the relatively low KM of 23 ± 4 μM together suggested that this enzyme acts uniquely on biuret physiologically. This is supported by the fact that of the 34 substrate analogs of biuret tested, only two demonstrated reactivity, both at less than 5% of the rate determined for biuret. Biuret hydrolase does not react with carboxybiuret, the product of the enzyme immediately preceding biuret hydrolase in the metabolic pathway for cyanuric acid. This suggests an unusual metabolic strategy of an enzymatically-produced intermediate undergoing non-enzymatic decarboxylation to produce the substrate for the next enzyme in the pathway.

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

“…This suggests that the diaminophosphate is labile, releasing an ammonia in an aqueous solution (consistent with previous reports) (1).…”

“…Previously, AtzE had been reported to be a biuret hydrolase (1,5,14,19), largely through comparison with the non-homologous biuret hydrolase of Rhizobium sp. (Table 1).…”

“…The presence of anthropogenic compounds in the environment provides selection pressures that can lead to the evolution of new metabolic pathways (1). One of the most well studied examples of an evolutionary response by bacterial to the presence of synthetic xenobiotics is that of the s-triazines (2).…”

“…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). It was therefore thought that the proceeding catabolic steps consisted of the deamination of biuret by AtzE to form allophanate, and allophanate deamination by AtzF to form carbamic acid.…”

“…AtzF is sometimes substituted by the homologous TrzF found in Enterobacter cloacae strain 99 (70 % identity to AtzF; (14)) and AtzE has been replaced with the non-homologous biuret amidohydrolase (BiuH, a (/)8 TIM barrel cysteine hydrolase) in Rhizobium leguminasorum bv. viciae 3841 (Table 1) (1,19).…”