Single-particle analysis of urea amidolyase reveals its molecular mechanism.

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.