Urea amidolyase (UA), a bifunctional enzyme that is widely distributed in bacteria, fungi, algae, and plants, plays a pivotal role in the recycling of nitrogen in the biosphere. Its substrate urea is ultimately converted to ammonium, via successive catalysis at the C-terminal urea carboxylase (UC) domain and followed by the N-terminal allophanate hydrolyse (AH) domain. Although our previous studies have shown that Kluyveromyces lactis UA (KlUA) functions efficiently as a homodimer, the architecture of the full-length enzyme remains unresolved. Thus how the biotin carboxyl carrier protein (BCCP) domain is transferred within the UC domain remains unclear. Here we report the structures of full-length KlUA in its homodimer form in three different functional states by negatively-stained single-particle electron microscopy. We report here that the ADP-bound structure with or without urea shows two possible locations of BCCP with preferred asymmetry, and that when BCCP is attached to the carboxyl transferase domain of one monomer, it is attached to the biotin carboxylase domain in the second domain. Based on this observation, we propose a BCCP-swinging model for biotin-dependent carboxylation mechanism of this enzyme.

中文翻译：

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尿素酰胺酶的单颗粒分析揭示了其分子机制。

尿素酰胺水解酶（UA）是一种双功能酶，广泛分布于细菌，真菌，藻类和植物中，在生物圈中的氮循环中起着关键作用。它的底物尿素通过在C端尿素羧化酶（UC）结构域的连续催化作用和随后的N端脲基甲酸酯水解（AH）结构域的催化作用最终转化为铵。尽管我们以前的研究表明乳酸克鲁维酵母UA（KlUA）作为同型二聚体有效发挥功能，但全长酶的结构仍未解决。因此，还不清楚如何在UC域内转移生物素羧基载体蛋白（BCCP）域。在这里，我们通过负染色的单粒子电子显微镜报道了其三聚体形式的全长KlUA在三种不同功能状态下的结构。我们在这里报告有或没有尿素的ADP结合结构显示了BCCP的两个可能的位置，具有优选的不对称性，并且当BCCP连接到一个单体的羧基转移酶结构域时，它连接到第二个结构域的生物素羧化酶结构域。基于此观察，我们提出了该酶的生物素依赖性羧化机理的BCCP摇摆模型。