Shapir Nir, Cheng Gang, Sadowsky Michael J, Wackett Lawrence P
Department of Biochemistry, Molecular Biology and Biophysics, 140 Gortner Lab, 1479 Gortner Ave., University of Minnesota, St. Paul, MN 55108, USA.
Appl Environ Microbiol. 2006 Apr;72(4):2491-5. doi: 10.1128/AEM.72.4.2491-2495.2006.
TrzF, the allophanate hydrolase from Enterobacter cloacae strain 99, was cloned, overexpressed in the presence of a chaperone protein, and purified to homogeneity. Native TrzF had a subunit molecular weight of 65,401 and a subunit stoichiometry of alpha(2) and did not contain significant levels of metals. TrzF showed time-dependent inhibition by phenyl phosphorodiamidate and is a member of the amidase signature protein family. TrzF was highly active in the hydrolysis of allophanate but was not active with urea, despite having been previously considered a urea amidolyase. TrzF showed lower activity with malonamate, malonamide, and biuret. The allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, was also shown to hydrolyze biuret slowly. Since biuret and allophanate are consecutive metabolites in cyanuric acid metabolism, the low level of biuret hydrolase activity can have physiological significance. A recombinant Escherichia coli strain containing atzD, encoding cyanuric acid hydrolase that produces biuret, and atzF grew slowly on cyanuric acid as a source of nitrogen. The amount of growth produced was consistent with the liberation of 3 mol of ammonia from cyanuric acid. In vitro, TrzF was shown to hydrolyze biuret to liberate 3 mol of ammonia. The biuret hydrolyzing activity of TrzF might also be physiologically relevant in native strains. E. cloacae strain 99 grows on cyanuric acid with a significant accumulation of biuret.
来自阴沟肠杆菌99株的脲基甲酸酯水解酶TrzF被克隆，在伴侣蛋白存在的情况下进行过表达，并纯化至同质。天然TrzF的亚基分子量为65,401，亚基化学计量比为α(2)，且不含大量金属。TrzF受苯基磷二酰胺的时间依赖性抑制，是酰胺酶特征蛋白家族的成员。TrzF在脲基甲酸酯水解方面具有高活性，但对尿素无活性，尽管它之前被认为是一种尿素酰胺水解酶。TrzF对丙二酸酯、丙二酰胺和缩二脲的活性较低。来自假单胞菌属菌株ADP的脲基甲酸酯水解酶AtzF也被证明能缓慢水解缩二脲。由于缩二脲和脲基甲酸酯是氰尿酸代谢中的连续代谢产物，缩二脲水解酶的低活性可能具有生理意义。一种含有编码产生缩二脲的氰尿酸水解酶的atzD和atzF的重组大肠杆菌菌株，以氰尿酸作为氮源时生长缓慢。产生的生长量与从氰尿酸中释放3摩尔氨一致。在体外，TrzF被证明能水解缩二脲以释放3摩尔氨。TrzF的缩二脲水解活性在天然菌株中可能也具有生理相关性。阴沟肠杆菌99株在氰尿酸上生长时会大量积累缩二脲。
