Department of Biochemistry, Biophysics & Molecular Biology, University of Minnesota, Minneapolis, Minnesota, USA; BioTechnology Institute, University of Minnesota, St Paul, Minnesota, USA.
Department of Biochemistry, Biophysics & Molecular Biology, University of Minnesota, Minneapolis, Minnesota, USA; BioTechnology Institute, University of Minnesota, St Paul, Minnesota, USA.
J Biol Chem. 2021 Jan-Jun;296:100055. doi: 10.1074/jbc.RA120.015631. Epub 2020 Dec 1.
Triuret (carbonyldiurea) is an impurity found in industrial urea fertilizer (<0.1% w/w) that is applied, worldwide, around 300 million pounds each year on agricultural lands. In addition to anthropogenic sources, endogenous triuret has been identified in amoeba and human urine, the latter being diagnostic for hypokalemia. The present study is the first to describe the metabolic breakdown of triuret, which funnels into biuret metabolism. We identified the gene responsible for triuret decomposition (trtA) in bacterial genomes, clustered with biuH, which encodes biuret hydrolase and has close protein sequence homology. TrtA is a member of the isochorismatase-like hydrolase (IHL) protein family, similarly to BiuH, and has a catalytic efficiency (kK) of 6 x 10 Ms, a K for triuret of 20 μM, and exquisite substrate specificity. Indeed, TrtA has four orders of magnitude less activity with biuret. Crystal structures of TrtA in apo and holo form were solved and compared with the BiuH structure. The high substrate selectivity was found to be conveyed by second shell residues around each active site. Mutagenesis of residues conserved in TrtA to the alternate consensus found in BiuHs revealed residues critical to triuret hydrolase activity but no single mutant evolved more biuret activity, and likely a combination of mutations is required to interconvert between TrtA, BiuH functions. TrtA-mediated triuret metabolism is relatively rare in recorded genomes (1-2%), but is largely found in plant-associated, nodulating, and endophytic bacteria. This study suggests functions for triuret hydrolase in certain eukaryotic intermediary processes and prokaryotic intermediary or biodegradative metabolism.
三聚脲（碳酰二脲）是工业尿素肥料中的一种杂质（<0.1%w/w），每年在世界各地约有 3 亿磅被应用于农业用地。除了人为来源外，内源性三聚脲已在变形虫和人尿中被发现，后者是低钾血症的诊断标志。本研究首次描述了三聚脲的代谢分解途径，其可流入缩二脲代谢途径。我们在细菌基因组中鉴定了负责三聚脲分解的基因（trtA），该基因与编码缩二脲水解酶的 biuH 聚类，且具有密切的蛋白质序列同源性。TrtA 是异羟肟酸酯水解酶（IHL）蛋白家族的成员，与 BiuH 相似，其催化效率（kK）为 6 x 10 Ms，对三聚脲的 K 值为 20 μM，具有极高的底物特异性。事实上，TrtA 对缩二脲的活性要低四个数量级。我们解析了 apo 和 holo 形式的 TrtA 晶体结构，并与 BiuH 结构进行了比较。发现高底物选择性是由每个活性位点周围的第二壳层残基传递的。将 TrtA 中保守残基突变为 BiuH 中的替代共识残基，揭示了对三聚脲水解酶活性至关重要的残基，但没有单个突变体进化出更多的缩二脲活性，可能需要突变的组合才能在 TrtA 和 BiuH 之间进行功能转换。在记录的基因组中，TrtA 介导的三聚脲代谢相对较少（1-2%），但主要存在于与植物相关、结瘤和内生的细菌中。本研究表明，三聚脲水解酶在某些真核生物中间过程和原核生物中间或生物降解代谢中具有功能。
