Cross-talk and ammonia channeling between active centers in the unexpecteddomain arrangement of glutamate synthase

Introduction: The complex iron-sulfur flavoprotein glutamate synthase catal yses the reductive synthesis of L-glutamate from 2-oxoglutarate and L-gluta mine, a reaction in the plant and bacterial pathway for ammonia assimilatio n. The enzyme functions through three distinct active centers carrying cut L-glutamine hydrolysis, conversion of 2-oxoglutarate into L-glutamate, and electron uptake from an electron donor. Results: The 3.0 Angstrom crystal structure of the dimeric 324 kDa core pro tein of a bacterial glutamate synthase was solved by the MAD method, using the very weak anomalous signal of the two 3Fe-4S clusters present in the as ymmetric unit. The 1472 amino acids of the monomer fold into a four-domain architecture. The two catalytic domains have canonical Ntn-amidotrans-feras e and FMN binding (beta/alpha)(8) barrel folds, respectively. The other two domains have an unusual "cut (beta/alpha)(8) barrel" topology and an unexp ected novel beta -helix structure. Channeling of the ammonia intermediate i s brought about by an internal tunnel of 31 Angstrom length, which runs fro m the site of L-glutamine hydrolysis to the site of L-glutamate synthesis. Conclusions: The outstanding property of glutamate synthase is the ability to coordinate the activity of its various functional sites to avoid wastefu l consumption of L-glutamine. The structure reveals two polypeptide segment s that connect the catalytic centers and embed the ammonia tunnel, thus bei ng ideally suited to function in interdomain signaling. Depending on the en zyme redox and ligation states, these signal-transducing elements may affec t the active site geometry and control ammonia diffusion through a gating m echanism.