FBS Colloquia No.270Department of Biomolecular Science and Reaction, ISIR-SANKEN (Kuroda lab.)

Neutron crystallography of copper amine oxidase: Novel hydrogen-bound and protonated structure and dynamic equilibrium of the active site

Enzymes are bio nanomachines that efficiently facilitate a number of chemical reactions under normal temperature and pressure conditions to substantially sustain biological phenomenon. They conduct metabolism and biosynthesis of biomolecules essential for living things. Our study aims to elucidate the molecular mechanism of enzymes through various structural biology techniques. We are focusing to experimentally determine the positions of lightest atom, hydrogen (or proton). Hydrogen atoms occupy about half number of all atoms in the protein and their movements play a key role in enzyme catalytic reactions. However, the positions of hydrogen atoms were unable to be easily determined by X-ray crystallography. This seminar will introduce recent topics of our study for bacterial copper amine oxidase including the neutron crystallography. Taking advantage that neutron crystallography visualizes hydrogen and deuterium atoms in the protein, we revealed unique active-site structures in CAO, including a keto/enolate equilibrium of the cofactor with a non-planar quinone ring, and quantum mechanical proton sharing between the cofactor. In addition, we succeeded X-ray crystallography under the temperature-controlled and unfrozen conditions using Humid Air and Glue-coating method. This technique exhibits the temperature-dependent equilibrium among reaction intermediates of CAO requiring the conformational changes of the active-site residues and cofactor. We believe that these researches could be the first step to grasp real state of the bio nanomachine, enzyme.