Tassoulas, Lambros2020-08-252020-08-252019-08https://hdl.handle.net/11299/215038University of Minnesota M.S. thesis.August 2019. Major: Microbial Engineering. Advisor: Lawrence Wackett. 1 computer file (PDF); vii, 65 pages.Triuret (carbonyl diurea) is an impurity of industrial urea fertilizer (<0.2%) which results in several hundred thousand tons applied, worldwide, each year on agricultural lands. Triuret has been described in the literature as early as 1870 when it was first synthesized and, although the natural source of triuret is unknown, it is hypothesized to be from oxidative uric acid metabolism. The biodegradation of triuret was known but, prior to this work, no enzymes had been identified. The triuret decomposing enzyme (TrtA) was discovered by observing two paralogs in gene clusters, one of which was well characterized as a biuret hydrolase (BiuH). TrtA, an amidohydrolase from the isochorismatase hydrolase like superfamily (IHL), similarly to BiuH, has a catalytic efficiency (kcat/Km) of 6x10^5 (M-1s-1) and a Km for triuret in the μM range with narrow substrate specificity. Crystal structures of TrtA in apo and holo form were solved which show an intriguing comparison with the BiuH structure where second shell residues around the near-identical active sites direct the specificity for each native substrate. With this discovery, the regulation of the triuret degradation operon, which can direct the mineralization of triuret completely to ammonium and carbon dioxide, was investigated in Herbaspirillum} sp. BH-1. The operon was shown to be specifically induced by biuret or triuret and suppressed in nitrogen sufficient conditions. Interestingly, the triuret hydrolase genes in the RefSeq database (<1%) come largely from plant-associated bacteria that can nodulate or are known endophytes and the broader context of this metabolism remains to be seen. As metabolism of triuret is relatively rare, and triuret is far less soluble in water than urea or ammonium nitrate, triuret may be considered a potential slow release N fertilizer.encarboxybiuretherbaspirillumnitrogen fertilizertriurettrtAtrtBThesis or Dissertation