The glutamine amidotranferases (GATs) are responsible for the hydrolysis of glutamine to produce ammonia for a diverse array of biosynthetic pathways. Thus far, there are 16 known GATs, all of which are modular and classified by their glutaminase domains. These domains are from four different ancestral groups: N-terminal nucleophile GATs (Ntn), Triad GATs, amidase GAT and Nitrilase GAT. In addition to their glutaminase domains, GATs have a second active site within their synthase domains. In this active site the ammonia produced from the glutaminase domain is combined with acceptor substrates for the synthesis of amino acids, purine and pyrimidine nucleotides, amino sugars and coenzymes. The progression of catalysis from the glutaminase domain to the synthase domain is highly regulated by substrate binding and the mechanism by which this regulation is achieved is GAT dependent. Given their importance in biosynthetic pathways, they are targets for potential antibiotic and anticancer therapies. Furthermore, they provide a model to study multi-step catalysis with a hierarchy of regulation. In this study we are interested in investigating the structural changes induced by either binding of the acceptor substrates or binding of a non-substrate small molecule and which specific residues propagate the conformational changes that are essential to the mechanism. Although all GATs share the ability to hydrolyze glutamine, their synthetase domains vary. This variation has lead to variations in how each GAT responds to the binding of their substrates. To expose which residues are critical to GATs from different ancestral groups three different GATs (pyridoxial 5'phosphate synthase from G. stearothemophilus, and GatCAB and cytosine triphosphate synthetase from A. aeolius) will be co-crystallized with their substrates in order to capture snapshots of their mechanisms.
In aim 1 an inactive PLP synthase mutant will be co-crystallized with its substrates. It is anticipated that this inactivating mutation will order the C-terminal tail of the PdxS subunit. A region that is essential to the complex's function. This will demonstrate how multi-subunit GATs communicate between subunits.
Aim 2 will focus on the role of the two divalent metals bound within GatCAB and how these metals are used to spatially orient all its substrates.
Aim 3 will identify why CTP synthetase uses a small molecule, GTP, instead of its substrates to induce conformational changes that enhance activity. CTP synthetase from A. aeolius is unique compared with its homologs from other organisms due to its significantly higher affinity for GTP. Understanding the mechanism behind these complex enzymes will provide a paradigm for structural studies for other multi-modular systems.

Public Health Relevance

Glutamine amidotransferases (GATs) are large, diverse modular proteins associated with many biologically important pathways. The goal of this project is to elucidate the conformational changes induced by substrate binding or the binding of a small molecule within this family of protein complexes. This will provide insight into the mechanisms behind modular proteins with specific levels of regulation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31GM093461-03
Application #
8390478
Study Section
Special Emphasis Panel (ZRG1-IMST-D (29))
Program Officer
Gaillard, Shawn R
Project Start
2010-12-01
Project End
2014-11-30
Budget Start
2012-12-01
Budget End
2013-11-30
Support Year
3
Fiscal Year
2013
Total Cost
$33,207
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109