Biocatalysis and Synthetic Biology Team, CSIRO Land & Water, Canberra, ACT, Australia.
Research School of Chemistry, Australian National University, Canberra, ACT, Australia.
PLoS One. 2018 Nov 6;13(11):e0206949. doi: 10.1371/journal.pone.0206949. eCollection 2018.
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.
三聚氰胺酸是一种常见的环境污染物，也是三嗪类化合物（包括莠去津和其他除草剂）分解代谢的中间产物。三聚氰胺酸通过多种细菌途径分解代谢，包括首先在假单胞菌 ADP 菌株中鉴定出的途径，该途径由一个位于可自我转移质粒上的单一、五个基因操纵子（atzDGEHF）编码。2018 年报道了其中两个基因（atzG 和 atzH）的发现，尽管确定了 atzG 的功能，但 atzH 的作用尚不清楚。在这里，我们首次在体外重建了完整的五蛋白三聚氰胺酸代谢途径，表明 AtzH 可能是一种酰胺酶，负责将 1,3-二羧基脲（AtzE 产物）转化为异氰尿酸（AtzF 底物）。我们已经解决了 AtzH 结构（NTF2 超家族的 DUF3225 蛋白），并利用它来预测底物结合口袋。定点突变实验表明，两个残基（Tyr22 和 Arg46）是催化所必需的。我们还表明，atzH 同源物在与 atzG 和 atzE 基因同源物相关的变形杆菌中普遍存在。这些 atzG-atzE-atzH 簇的遗传背景表明它们在含氮化合物的代谢中起作用。此外，它们在许多基因组中的存在而没有 atzD 和 atzF 的同源物表明，atzG-atzE-atzH 簇可能先于三聚氰胺酸代谢操纵子的进化而存在。
