title: Purification and characterization of TrzF: biuret hydrolysis by allophanate hydrolase supports growth.
abstract: 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.
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, 140 Gortner Lab, 1479 Gortner Ave., University of Minnesota, St. Paul, MN 55108, USA.
authors.forename: Nir
authors.initials: N
authors.lastname: Shapir
authors.forename: Gang
authors.initials: G
authors.lastname: Cheng
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sadowsky
authors.forename: Lawrence P
authors.initials: LP
authors.lastname: Wackett
mesh_terms: D000492:Allophanate Hydrolase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001737:Biuret / Q000378:metabolism; D003470:Culture Media; D016972:Enterobacter cloacae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D006868:Hydrolysis; D014227:Triazines / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D014227:Triazines; D001737:Biuret; D000581:Amidohydrolases; D000492:Allophanate Hydrolase; C067141:cyanuric acid amidohydrolase; C004632:cyanuric acid