Mechanistic Insights into the Functioning of a Two-Subunit GMP Synthetase, an Allosterically Regulated, Ammonia Channeling Enzyme

Abstract

Guanosine 5′-monophosphate (GMP) synthetases, enzymes that catalyze the conversion of xanthosine 5′-monophosphate (XMP) to GMP, are composed of two different catalytic units, which are either two domains of a polypeptide chain or two subunits that associate to form a complex. The glutamine amidotransferase (GATase) unit hydrolyzes glutamine generating ammonia, and the ATP pyrophosphatase (ATPPase) unit catalyzes the formation of an AMP-XMP intermediate. The substrate-bound ATPPase allosterically activates GATase, and the ammonia thus generated is tunneled to the ATPPase active site where it reacts with AMP-XMP generating GMP. In ammonia channeling enzymes reported thus far, a tight complex of the two subunits is observed, while the interaction of the two subunits of Methanocaldococcus jannaschii GMP synthetase (MjGMPS) is transient with the underlying mechanism of allostery and substrate channeling largely unclear. Here, we present a mechanistic model encompassing the various steps in the catalytic cycle of MjGMPS based on biochemical experiments, crystal structure, and cross-linking mass spectrometry guided integrative modeling. pH dependence of enzyme kinetics establishes that ammonia is tunneled across the subunits with the lifetime of the complex being ≤0.5 s. The crystal structure of the XMP-bound ATPPase subunit reported herein highlights the role of conformationally dynamic loops in enabling catalysis. The structure of MjGMPS derived using restraints obtained from cross-linking mass spectrometry has enabled the visualization of subunit interactions that enable allostery under catalytic conditions. We integrate the results and propose a functional mechanism for MjGMPS detailing the various steps involved in catalysis.

Understanding structural and functional diversity of ATP-PPases using protein domains and functional families in CATH database

“…Drawing on the enriched structural information and a range of bioinformatic tools, we performed detailed classification and characterization of the GMPS and NADS FunFams. Structural comparisons between four GMPS FunFams showed that the Lid region is differentially conserved between FunFams, supported by previous experiments 7,27,28 . I n addition, sequence comparisons between GMPS FunFams exposed several residues within the Lid region that are differentially conserved, providing new insights into various modes by which the Lid region regulates Gln-hydrolysis and NH 3 -transportation.…”

“…Although lacking the intrachain ATP-PPase-GATase interaction, the archaeal MDA is similar to the bacterial one ( Figure.3a ). The predicted archaeal GMPS complex with the GATase bound to the ATP-PPase subunit is similar to the unrotated Plasmodium GMPS 27 . Since archaeal GMPS FunFam have hydrophobic and aromatic residues conserved on SDPs 222, 370 and 415 similar to the Plasmodium GMPS FunFam ( Table.3 ), the bound archaeal GATase subunit may undergo further rotation and activation like the Plasmodium GMPS 27 .…”

“…The predicted archaeal GMPS complex with the GATase bound to the ATP-PPase subunit is similar to the unrotated Plasmodium GMPS 27 . Since archaeal GMPS FunFam have hydrophobic and aromatic residues conserved on SDPs 222, 370 and 415 similar to the Plasmodium GMPS FunFam ( Table.3 ), the bound archaeal GATase subunit may undergo further rotation and activation like the Plasmodium GMPS 27 . The key difference between archaeal GMPS ATP-PPase domain and the other GMPS is the SDP residue R/W373.…”

“…The residues D372, E375, K389, H391 and H392 7,28,48 are involved in the first step of GMP production ( Equation.1 ), which is adenylating the substrate XMP to generate XMP-AMP 2,49 . However, these catalytic residues are distant from the XMP-binding site in all the resolved GMPS structures 7,27,28,46,47 . This suggests a potential common function of the Lid region in delivering the catalytic residues D372, E375 and the Lid motif to the ATP-PPase domain catalytic site 7,16,46,48 .…”

“…The exceptions are all from the archaeal GMPS FunFam (FunFam208), where the ATP-PPase and GATase subunits exist as separate polypeptides. These subunits transiently interact with each other to form a holoenzyme 27,47 . As a result, the Lid region of the apo-form archaeal ATP-PPase subunits (residues 132-153 on the representative structure, PDB:3a4i) is exposed on the surface of the ATP-PPase domain, resulting in low resolution and Zebra3D subclassification as a separate subgroup ( Figure.2b, Table.2 ).…”