Microbial biodegradation of biuret: defining biuret hydrolases within the isochorismatase superfamily

Abstract

Biuret is a minor component of urea fertilizer and an intermediate in s-triazine herbicide biodegradation. The microbial metabolism of biuret has never been comprehensively studied. Here, we enriched and isolated bacteria from a potato field that grew on biuret as a sole nitrogen source. We sequenced the genome of the fastest-growing isolate, Herbaspirillum sp. BH-1 and identified genes encoding putative biuret hydrolases (BHs). We purified and characterized a functional BH enzyme from Herbaspirillum sp. BH-1 and two other bacteria from divergent phyla. The BH enzymes reacted exclusively with biuret in the range of 2-11 µmol min mg protein. We then constructed a global protein superfamily network to map structure-function relationships in the BH subfamily and used this to mine > 7000 genomes. High-confidence BH sequences were detected in Actinobacteria, Alpha- and Beta-proteobacteria, and some fungi, archaea and green algae, but not animals or land plants. Unexpectedly, no cyanuric acid hydrolase homologs were detected in > 90% of genomes with BH homologs, suggesting BHs may have arisen independently of s-triazine ring metabolism. This work links genotype to phenotype by enabling accurate genome-mining to predict microbial utilization of biuret. Importantly, it advances understanding of the microbial capacity for biuret biodegradation in agricultural systems.

Discovery of an ultraspecific triuret hydrolase (TrtA) establishes the triuret biodegradation pathway

“…The triuret hydrolase enzyme, TrtA, from the Herbaspirillum species shows 48% amino acid sequence identity to the previously crystallized BiuH from Rhizobium leguminosarum b.v viciae 3841 (NCBI accession no. CAK10578.1) (9,10). Both species have operons that contain TrtA, BiuH and AtzF that combine to support the growth of the bacteria by liberating all four nitrogens from triuret as assimilable ammonia (Supp.…”

“…A generalist enzyme (RhdBiuH), with 67% sequence identity to BiuH, was found in the genome of Rhodococcus sp. Mel which is co-located with a cyanuric acid hydrolase (AtzD) and has no triuret hydrolase in the genome (9). The enzyme hydrolyzes biuret at a relatively slow rate (1.7 U/mg) compared to true BiuHs (>10 U/mg) and has a specific activity for triuret of 0.3 U/mg which is 25 fold higher than BiuH (Table 4).…”

“…A similar compound to triuret, biuret (carbamylurea) with one less carbamoyl group, has a known biodegradation pathway through biuret hydrolase (E.C 3.5.1.84) (9,10). Biuret hydrolase (BiuH) hydrolyzes biuret to allophanate and is part of the Isochorismatase-like hydrolase (IHL) protein family within the cysteine hydrolase superfamily (10,11).…”

“…Prior to this study, the only known precursor of biuret was cyanuric acid, a metabolite of s-triazine compounds mostly comprised of herbicides (Figure 1). However, based on bioinformatics analysis, biuret metabolism appears to be mostly independent of cyanuric acid metabolism (9,10). In lieu of the cyanuric acid hydrolase (AtzD, E.C 3.5.2.15) not being in an operon with BiuH, the genome contexts of several hundred BiuH encoding sequences revealed a cooccurring homologous sequence, TrtA, with ~50% amino acid sequence identity on average but showing no activity on biuret.…”

A novel decarboxylating amidohydrolase involved in avoiding metabolic dead ends during cyanuric acid catabolism in Pseudomonas sp. strain ADP

“…viciae 3841 pathway employs cyanuric acid amidohydrolase (E.C. 3.5.2.15)[ 2 , 6 , 7 ] to ring-open cyanuric acid and the product (1-carboxybiuret) then undergoes solvent-mediated hydrolysis under physiological conditions to form biuret. Biuret is deaminated by biuret amidohydrolase (E.C.…”