A 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 k(cat)/K(M) of 1.7 × 10(5) M(-1)s(-1) and the relatively low K(M) 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.

Figure 1

Alignments of gene regions encoding functional cyanuric acid hydrolases from various soil bacteria.

Fig. 2

Amino acid sequence alignment in region of superfamily active site cysteine for biuret hydrolase (BH) with: Streptococcus pneumoniae nicotinamidase (3o90), Thermoplasma acidophylum N-carbamoyl-sarcosine amidase Ta0454 (3EEF), and Pyrococcus horikoshi pyrazinamidase (1IM5), for which X-ray structures are known. An * indicates a completely conserved residue and a: indicates a highly conserved residue. The active site cysteine is highlighted with a box.

Figure 2

¹³C-NMR of enzyme and non-enzyme formed products. (A) [U-¹³C]-Biuret reacted with biuret hydrolase. Allophanate peaks were observed at 1.5 hrs. (B) Reaction mixture in (A) allowed to stand at 20°C for 67.5 h. (C) Treatment of reaction mixture in (B) with urease. Urea peak undetectable after 1.25 h. NMR acquisition times vary.

Figure 3

Disappearance of [U-13C]-carboxybiuret and appearance of [U-13C]-bicarbonate in the absence (A) or presence (B) of biuret hydrolase.

Scheme 1

Biuret is an intermediate in the metabolism of s-triazine compounds and could be metabolized to allophanate or urea.

Scheme 2

Hypothetical modes of C-N bond cleavage by biuret hydrolase demonstrating all lead to formation of HCO₃⁻.