The Conformational Change Mechanism Of Urea Amidolyase Function Analyzed By Single Particle Electron Microscopy

Urea amidolyase(UA),a member of the biotin-dependent carboxylase family,plays an important role in recycling nitrogen by catalyzing the conversion of urea to ammonium.it is a bifunctional enzyme that widely distributed in bacteria,fungi,algae and plants.The conversion procedure performs consecutively at the C-terminal biotin carboxylase(BC),carboxyltransferase(CT)and the N-terminal allophanate hydrolase domain corresponding to carboxylating biotin,transferring the carboxyl to urea and hydrolyzing the intermediate product into ammonium respectively.The whole process needs tight communications between individual domains for transferring the substrate/product for related catalytic activities.However interestingly,the other members included in the family have to function as oligomers,while it's not necessary for UA to be active.Although each terminus of the sequence has been crystalized successively,the C-terminus' only captured the CT-BCCP interaction state,the full-length architecture of the enzyme remains unresolved due to the unsuccessful crystallization,which makes the overall molecular architecture of the full-length UA still unavailable hence the mechanism of long-term conformational change in mediating communications between individual domains remains unclear.In the preliminary work,we have obtained highly uniform holoenzyme of Urea amidolyase of kluyveromyces lactis.In this paper,the overall structure and conformational changes of KIUA soluble homologous dimer were reported by single particle transmission electron microscopy for the first time.Interestingly,regardless of incubating with or without ligand and substrate,the defined structures all represent an intermediate state that the biotin-carboxyl carrier protein(BCCP)domain anchoring alternatively at either BC or CT domain of individual monomer,which delineate a possible synergic mechanism of the individual monomer thereafter explains its more efficient activity in the homodimer form.Furthermore,we may conclude that the BCCP domain swinging between BC and CT domains to complete the carboxylation of biotin and transfer of carboxyl to urea is most likely in an energy-saving and random translocation mode.