Structural Analysis of Mechanism and Regulation of Glutamine Amidotransferases.

Abstract

Glutamine amidotransferases (GATs) use glutamine as a nitrogen source for a diverse array of biosynthetic processes. GATs hydrolyze glutamine in a glutaminase domain and transfer ammonia to various acceptor substrates in a synthase domain. Allosteric regulation plays a large role in the mechanisms of GATs. Substrate binding induces structural changes to both the synthase and glutaminase domains, contributing to product formation. Capturing these structural changes is a challenge in understanding the mechanisms of GATs. This thesis investigates two different GATs: pyridoxal 5’-phosphate synthase (PLPS) from the bacterium Geobacillus stearothemophilus and cytosine triphosphate synthetase (CTPS) from the bacterium Aquifex aeolicus. PLPS generates pyridoxal 5’-phosphate (PLP) from glutamine, ribose 5-phosphate (R5P) and glyceraldehyde 3-phosphate (G3P). The mechanism of PLP formation was probed by solving crystal structures in three distinct states that capture two covalent intermediates of the synthase active site and one of the glutaminase active site. The structures reveal a complex set of conformational changes that sequester the active sites from bulk solvent. Key roles were identified for several charged amino acids. CTPS converts uridine triphosphate (UTP) to cytosine triphosphate (CTP) through ATP-activated amination of UTP. GTP serves as an allosteric activator of CTPS, increasing glutaminase activity 3-4 fold when glutamine is the nitrogen source. To investigate the GTP binding site I used X-ray crystallography, negative-stain electron microscopy and binding experiments to monitor the structural changes induced by GTP. The basis for CTPS inhibition by excessive GTP was established.