Alice Grieco
CSIC
ABSTRACT
Human NQO1 is a flavoenzyme essential for the antioxidant defense system, stabilization of tumor suppressors, and activation of quinone-based chemotherapeutics. NQO1 is over-expressed in tumors such as thyroid, breast, and lung, which makes this enzyme an attractive cancer target for drug discovery. NQO1 exists as a homodimer, in which the N-terminal domain binds FAD while binding of NADH, substrates, and competitive inhibitors, also require the smaller C-terminal domain. NQO1 develops its catalytic function by the “ping-pong” mechanism consisting of two ordered steps: 1) Binding and oxidation of the electron donor NADH by FAD that reduces to FADH2 with NAD+ subsequently leaving the binding site; 2) Binding and reduction of a quinone substrate (e.g. menadione) by the FADH2. Crystallographic structures of NQO1 with and without NADH have demonstrated that NQO1 undergoes local conformational changes in which the active site opens and closes during the catalytic mechanism.
In the attempt to decipher the complex catalytic mechanism of human NQO1, we have recently carried out initial serial femtosecond crystallography (SFX) experiments at the LCLS. The experiments were conducted at MFX instrument during beamtime LW79 in late May 2021. Microcrystals of NQO1 (10 x 2 x 2 µm3) were delivered to the X-ray FEL beam using a 3D-printed sample delivery device developed by the Ros group at Arizona State University. A total of 479,075 images were collected, of which 17,379 were found to be hits. Of those hits, 17,734 were successfully indexed, integrated, and merged. From these experiments, we have determined the SFX structure of NQO1 to 2.5 Å resolution. To further understand the catalytic mechanism of NQO1, time-resolved SFX experiments in the presence of the substrate NADH at various time delays are required. To this end, we have recently submitted an LCLS proposal for run 20 (LX81, PI: Jose M. Martin-Garcia). With the proposed experiments we aim at determining the structure of the intermediates involved in the oxidation process of NADH by NQO1.
Poster Session Link:
https://gather.town/invite?token=0pEoq7VP
If you have any questions for the presenter, please contact them by either one of the following ways:
Alice Grieco Community Page Link
Email: agrieco@iqfr.csic.es