Abstract
Cyanuric acid is a common environmental contaminant and a metabolic intermediate in the catabolism of s-triazine compounds, including atrazine and other herbicides. Cyanuric acid is catabolized via a number of bacterial pathways, including one first identified in Pseudomonas sp. strain ADP, which is encoded by a single, five-gene operon (atzDGEHF) found on a self-transmissible plasmid. The discovery of two of the five genes (atzG and atzH) was reported in 2018 and although the function of atzG was determined, the role of atzH was unclear. Here, we present the first in vitro reconstruction of the complete, five-protein cyanuric acid catabolism pathway, which indicates that AtzH may be an amidase responsible for converting 1,3-dicarboxyurea (the AtzE product) to allophanate (the AtzF substrate). We have solved the AtzH structure (a DUF3225 protein from the NTF2 superfamily) and used it to predict the substrate-binding pocket. Site-directed mutagenesis experiments suggest that two residues (Tyr22 and Arg46) are needed for catalysis. We also show that atzH homologs are commonly found in Proteobacteria associated with homologs of the atzG and atzE genes. The genetic context of these atzG-atzE-atzH clusters imply that they have a role in the catabolism of nitrogenous compounds. Moreover, their presence in many genomes in the absence of homologs of atzD and atzF suggests that the atzG-atzE-atzH cluster may pre-date the evolution of the cyanuric acid catabolism operon.
Highlights
The symmetrical triazine cyanuric acid (1,3,5-triazine-2,4,6-triol) is a common anthropogenic compound that is used in the synthesis of a variety of disinfectants and herbicides
AtzH facilitates the only previously uncontrolled step in the catabolic pathway, and as such, we believe that we have identified the last remaining enzyme in the Pseudomonas sp. strain ADP cyanuric acid catabolism pathway (Fig 1B)
Given the amidase function of AtzE and proposed amidase activity of AtzH, it is perhaps unsurprising that the atzG-atzE-atzH-like clusters tend to be associated with genes predicted to be involved in nitrogen compound metabolism
Summary
The symmetrical triazine cyanuric acid (1,3,5-triazine-2,4,6-triol) is a common anthropogenic compound that is used in the synthesis of a variety of disinfectants (e.g., trichlorocyanuric acid) and herbicides (e.g., atrazine). 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) undergoes solvent-mediated hydrolysis under physiological conditions to form biuret. Strain ADP cyanuric acid mineralization pathway is encoded by a single, five-gene operon: atzDGEHF [8]. Strain ADP ring opens cyanuric acid using cyanuric acid amidohydrolase (AtzD) [7,9] In this pathway, it is 1-carboxybiuret that is deaminated by the enzymatic step, which is catalyzed. Strain ADP is unable to degrade biuret or urea, and these solvent-mediated reactions effectively form metabolic ‘dead-ends’ [2,10]. We present the structure of AtzH and explore its potential function
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