Abstract
Urea is the degradation product of a wide range of nitrogen containing bio-molecules. Urea amidolyase (UA) catalyzes the conversion of urea to ammonium, the essential first step in utilizing urea as a nitrogen source. It is widely distributed in fungi, bacteria and other microorganisms, and plays an important role in nitrogen recycling in the biosphere. UA is composed of urea carboxylase (UC) and allophanate hydrolase (AH) domains, which catalyze sequential reactions. In some organisms UC and AH are encoded by separated genes. We present here structure of the Kluyveromyces lactis UA (KlUA). The structure revealed that KlUA forms a compact homo-dimer with a molecular weight of 400 kDa. Structure inspired biochemical experiments revealed the mechanism of its reaction intermediate translocation, and that the KlUA holo-enzyme formation is essential for its optimal activity. Interestingly, previous studies and ours suggest that UC and AH encoded by separated genes probably do not form a KlUA-like complex, consequently they might not catalyze the urea to ammonium conversion as efficiently.
Highlights
Degradation of a wide range of nitrogen-containing bio-molecules generates urea
If there is no covalent linkage between the allophanate hydrolase (AH) and urea carboxylase (UC) domains, the latter is not strong enough to hold them to form a stable complex. This suggests that AH and UC encoded by separated genes probably do not form a Kluyveromyces lactis urea amidolyase (UA) (KlUA)-like complex, consistent with studies on a number of such AH and UC proteins [16,18,32]
A continuous positively charged surface patch connects the active sites of the UC CT domain and the AH N domain in the same KlUA polypeptide, our data argue against a model that it mediates substrate channeling of allophanate
Summary
Degradation of a wide range of nitrogen-containing bio-molecules generates urea. Mammals do not directly utilize urea, and excrete it into the environment. Many plants, fungi and bacteria can utilize urea as a nitrogen source [1,2,3] They reintegrate its nitrogen into the biosphere, playing important roles in the nitrogen recycling within the biosphere. Weight of 400 kDa. Structure guided biochemical experiments provided insights into allophanate translocation between the UC and AH domains, and indicated that the KlUA holo-enzyme formation is required for its optimal activity. Previous studies and ours suggest that UC and AH encoded by separated genes probably do not form a KlUA-like complex. They probably catalyze the urea to ammonium conversion less efficiently
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