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Structure

GMP synthetase is a homodimer enzyme, in which each monomer is composed of 693 amino acids and weights 76,2 kDa ^[2]. Each monomer is composed of , encoded by a single gene: a N-terminal glutaminase domain (GATase domain), and a C-terminal synthetase domain.

Secondary structure of GMP synthetase

GTase domain

The GATase domain, stretched from residue 27 to residue 216, is composed of a single structural domain. It is constituted of a central β-sheet surrounded by several α-helices. It contains composed of residues Cys104, His190, and Glu192.

Synthetase domain

, stretched from residue 217 to residue 693, can be divided into three different sub-domains: an ATP pyrophosphatase domain (ATPPase domain) and two dimerization domains D1 and D2.

The ATPPase sub domain (residues 217-435) is composed of a 5-stranded parallele β-sheet-sandwiched between 9 α-helices.

The D1 sub-domain (residues 450-578), absent in bacteria and archaea, is involved in dimerization and substrate binding. It is constituted of an anti-parallel three-stranded β-sheet surrounded by five α-helices. The middle β-strand stretches out to form (Ile514–Tyr528) which interacts with the XMP binding site of the other subunit of the dimer.

The second dimerization sub-domain, D2 (residues 579-693) is similar to D1 sub-domain and they are superposed. This leads also to the D2 sub-domain to be superposed on the ATPPase sub-domain allowing the formation of the XMP active site in the synthetase domain.

is located between the ATPPase sub-domain and the D2 sub-domain. When XMP is bound to it, it is allostericly regulated and covered by a lid motif (residues 368-408) closing in over the active site. Thus, the XMP is wedged between a Pro-rich region (residues 438–441) and a loop (residues 383–385).

The synthetase domain binds several cofactors. Indeed, are bound to the ATPPase and D2 sub-domains. There are also Mg2+ and ATP which can bind.

There are 2 isoforms produced by alternative splicing: the isoform 1, which was just described, and the isoform 2. The sequence of this isoform differs from the first isoform by the missing sequence from residue 10 to residue 108. Thus, this isoform is only composed of 594 amino acids and weights 65,9 kDa ^[3].

Function

GMP

GMP synthetase is a cytosolic enzyme belonging to the glutamine amidotransferases family. These amidotransferases catalyse the amination of a wide range of molecules using the amide nitrogen of the side chain of glutamine. GMP synthetase is one of the three glutamine amidotransferases that plays a role in the de novo purine biosynthesis ^[4]. Indeed, thanks to its bifunctional two domains, GMP synthetase catalyses the final step in the de novo synthesis of GMP from XMP in the presence of other cofactors including ATP, glutamine and water. The global reaction is summarized below:

ATP + XMP + L-glutamine + H2O --> AMP + diphosphate + GMP + L-glutamate.

Actually, the enzyme operates in two successive steps:

1) L-glutamine + H2O --> L-glutamate + NH3
2) ATP + XMP + NH3 --> AMP + pyrophosphate + GMP

First, the glutaminase domain generates ammonia from glutamine-hydrolysis when L-glutamine binds to the catalytic triad. Then, an activation step prepares the XMP for amination. Indeed, GPM synthetase activates its XMP substrate by adenylylation on the xanthine C2 oxygen, which can then be primed for attack by a nitrogen nucleophile. In order to perform the second reaction, the glutamine-derived ammonia needs to be transferred to the . This one is located in the active site of the synthetase domain, situated no far away from the catalytic triad. The ammonia translocation is enabled by a channel between the two active sites. This channel is formed thanks to a conformational change of the catalytic triad, further to its production. Thus, the activated XMP is aminated to produce GMP. Then, the GMP is released and will be used as a monomer in RNA. ^[4]

Activation and inhibition

The presence of free Mg2+ is essential for activation of the GMP synthetase and a complex between ATP and Mg2+ can be formed but MgATP2− alone is not sufficient for catalysis. Moreover, the total chelation of free Mg2+ by ATP results of inactivation of the enzyme. ^[5]

Inhibitors

-Decoyinine is an uncompetitive inhibitor
-Inorganic pyrophosphate is the most effective inhibitor and it is competitive toward ATP
-Psicofuranin is known to inhibit this enzyme.
-6-Diazo-5-oxo-L-norleucine (DON) is a glutamine antagonist

-Acivicin is an analog of glutamine that irreversibly inhibits GMPS and other glutamine-dependant amidotransferases

Relevance and Medical implications

Cancer`

Several enzymes in the nucleotide metabolism display an increased activity in rapidly dividing cells due to an increased demand for nucleotides. One of the characteristic of cancer cells is their high division rate and proliferation. Thus, human GMPS is identified as a potential target for anti-cancer therapy. It was shown that inhibition of GMP synthethase by acivicin inhibited the growth of hepatoma cells in culture thanks to a depression of the GTP pool. After its discovery in 1972, acivicin was used as an anti-cancer agent, but trials were unsuccessful due to toxicity. ^[6] GMPS was also found upregulated in tumorigenic cells.

Immunosuppressive therapy

Proper nucleotide metabolism is an important factor for immune cell maturation and function. Thus, inherited defects in purine metabolism enzymes result in immunodeficiency in patients. Because of the importance of guanine nucleotide synthesis in immune cells functions, GMP synthetase is a potential target for immunosuppressive therapy. ^[7]

Acute myeloid leukemia

GMPS is also implied in acute myeloid leukemia. Translocations of the MLL (Mixed-Lineage Leukemia) gene at chromosome band 11q23 are recurrent in patients with leukemia after classical treatments. The MLL gene fuses with many different partner genes, most of which remain unknown. But in 2000, Pegram et al. identified GMPS gene to be a new partner of MLL. It was the first gene of this type and located on chromosme 3q to be found in leukemia-associated translocations. ^[8]

Anti-microbial/antifungal target

The fact that the human GMPS (hGMPS) differs from its bacterial and archaeal counterpart by having an additional dimerization sub-domain is exploited in the development of anti-bacterial or anti-parasitic drugs. Indeed, the drugs can be specific for bacterial or parasitic nucleotide metabolism, without affecting human cells function. For instance, a team showed that several drugs can inhibit the GMP synthetase activity in Candida albicans and Aspergillus fumigatus. Indeed, after incubation of a cell extract with an inhibitor of GMPS, GMPS activity was significantly reduced. GMP synthase activity is essential and required for virulence of both pathogens, thus constituting an interesting antifungal target. ^[9]

Histone deubiquitylation

An allosteric role of GMPS in gene regulation through histone deubiquitylation was recently discovered. GMPS interacts with the deubiquitinatin enzyme ubiquitin-specific protease 7 (USP7). Indeed, GMPS can allosterically stabilize the active conformation and promote the activation of the protein. Thus, it permits a precise regulation of USP7 expression and is therefore necessary for maintaining proper cell proliferation. Together with USP7, GMPS is involved in cell survival, chromatin maintenance and transcriptional regulation of target genes. ^[10]