Refined Crystal Structures of Unligated Adenylosuccinate Synthetase fromEscherichia coli

Silva, M.Mercedes; Poland, Bradley W.; Hoffman, Chad R.; Fromm, Herbert J.; Honzatko, Richard B.

doi:10.1006/jmbi.1995.0629

Cited by 40 publications

(45 citation statements)

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“…These missing residues corresponded to the flexible active site loops described in other species (41) , which only become ordered in these species when the enzyme is fully ligated.…”

Section: Crystal Structure Of Unligated Cryptococcal Adssmentioning

confidence: 99%

“…AdSS from E. coli is arguably the best characterized (41,97,98) and has 42% identity with the cryptococcal enzyme. Crystal structures are also available for the proteins from Homo sapiens (54%, PDB ID: 2V40); Mus musculus (54%, (50,99) ); the bacteria Yersinia pestis (42%, PDB ID: 3HID), Campylobacter jejuni (43%, PDB ID: 3R7T) and Burkholderia thailandensis (40%, PDB ID: 3UE9); the plants Arabidopsis thaliana (55%) and Triticum aestivum (53%, (49) ); the eukaryotic parasite Plasmodium falciparum (46% (48) ; and the extremophile archaeon…”

Section: An Existing Adss Inhibitor Exhibits Limited Antifungal Activitymentioning

confidence: 99%

“…This GTP and Mg 2+ dependent reaction proceeds via two steps: firstly, the γ-phosphate from GTP is transferred to 6-oxygen of IMP, forming the intermediate 6-phosphoryl IMP (6-PIMP); secondly, the 6-phosphoryl group is displaced by the α-amino group of aspartate to form adenylosuccinate (39) (Fig 9A). Structural studies in Escherichia coli and other organisms have shown that the enzyme forms a dimer, with each subunit contributing an arginine residue to the active site of the other molecule (40,41,47,48) . In the absence of its substrate, the active site of AdSS is disordered, however upon ligand binding, the substrate binding pocket and dimer formation are stabilized (44,94) .…”

Section: An Existing Adss Inhibitor Exhibits Limited Antifungal Activitymentioning

confidence: 99%

“…The first reported crystal structure of an AdSS enzyme was from Escherichia coli, and it remains the best characterised form of this enzyme (Fig 2) (40,41) . E. coli AdSS is a functional homodimer, with its structure comprised of a nine-stranded β-sheet backbone and four large subdomains.…”

Section: Figmentioning

confidence: 99%

“…The enzyme has been crystallized in the apo-form (41,50) as well as complexed with various combinations of IMP, GTP, GDP, aspartate, 6-PIMP, Mg2+, and two AdSS inhibitors: the anti-tumor aspartate analogue hadacidin (56,61) and the adenosine 5'-monophosphate-mimicking herbicide hydantocidin (63) .…”

Section: An Existing Adss Inhibitor Exhibits Limited Antifungal Activitymentioning

confidence: 99%

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Disruption of de novo ATP biosynthesis abolishes virulence in Cryptococcus neoformans

Blundell¹

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With the spread of the AIDS pandemic, opportunistic fungal pathogens like Cryptococcus neoformans are a growing cause of morbidity and mortality among immunocompromised populations worldwide. In part owing to a common eukaryotic ancestry that results in shared physiology between fungal and human cells, these fungal infections are difficult to treat. The limited antifungal agents currently available therefore tend to target the few key differences between the two systems to avoid inadvertently harming the host. To address the current paucity of antifungal therapeutic agents, further research into fungal-specific drug targets is required. Adenylosuccinate synthetase (AdSS) is a crucial enzyme in the ATP biosynthetic pathway, catalyzing the magnesium-and GTP-dependant formation of adenylosuccinate from inosine monophosphate and aspartate.Over the course of my project I have investigated the potential of this enzyme as an antifungal drug target. By deleting the AdSS-encoding ADE12 gene from the C. neoformans genome, I found that loss of AdSS function results in adenine auxotrophy in C. neoformans, as well as complete loss of virulence in a murine infection model. In contrast, deleting the APH1 gene, encoding the adenine salvage enzyme adenine phosphoribosyltransferase, had no deleterious effects on cell growth or virulence in the murine model. I expressed and purified Cryptococcal AdSS from Escherichia coli and optimised conditions to generate high-quality AdSS protein crystals. These were used to complete X-ray diffractions studies, allowing me to determine the crystal structure of the enzyme. The structure of the enzyme's unbound form was solved first, and then used to solve the structure of the enzyme ligated with IMP and GDP. These are the first examples of an AdSS crystal structure from a fungal species. Together with enzyme kinetic studies, this structural information allowed me to compare the fungal enzyme with the human ortholog. This revealed species-specific differences that are potentially exploitable via rational drug design. These results validate AdSS as a promising antifungal drug target and lay a foundation for future in silico and in vitro screens for novel antifungal compounds.3

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“…These missing residues corresponded to the flexible active site loops described in other species (41) , which only become ordered in these species when the enzyme is fully ligated.…”

Section: Crystal Structure Of Unligated Cryptococcal Adssmentioning

confidence: 99%

Section: An Existing Adss Inhibitor Exhibits Limited Antifungal Activitymentioning

confidence: 99%

Section: An Existing Adss Inhibitor Exhibits Limited Antifungal Activitymentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

Section: An Existing Adss Inhibitor Exhibits Limited Antifungal Activitymentioning

confidence: 99%

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Disruption of de novo ATP biosynthesis abolishes virulence in Cryptococcus neoformans

Blundell¹

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Leishmania donovani adenylosuccinate synthetase requires IMP for dimerization and organization of the active site

Mochi,

Jani,

Shah

et al. 2024

FEBS Letters

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Adenylosuccinate synthetase (AdSS), which catalyses the GTP‐dependent conversion of inosine monophosphate (IMP) and aspartic acid to succinyl‐AMP, plays a major role in purine biosynthesis. In some bacterial AdSS, it is implicated that IMP binding is important to organize the active site, but in certain plant AdSS, GTP performs this role. Here, we report that in Leishmania donovani AdSS, IMP binding favoured dimerization, induced greater conformational change and improved the protein stability more than GTP binding. IMP binding, which resulted in a network of hydrogen bonds, stabilized the conformation of active site loops and brought the switch loop to a closed conformation, which then facilitated GTP binding. Our results provide a basis for designing better inhibitors of leishmanial AdSS.

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Adenylosuccinate Syntheatase: Recent Developments

Honzatko

Stayton

Formm

1999

Advances in Enzymology - And Related Areas of Molecular Biology

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Refined Crystal Structures of Unligated Adenylosuccinate Synthetase fromEscherichia coli

Cited by 40 publications

References 0 publications

Disruption of de novo ATP biosynthesis abolishes virulence in Cryptococcus neoformans

Disruption of de novo ATP biosynthesis abolishes virulence in Cryptococcus neoformans

Leishmania donovani adenylosuccinate synthetase requires IMP for dimerization and organization of the active site

Adenylosuccinate Syntheatase: Recent Developments

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