Purification, crystallization and preliminary X-ray analysis of adenylosuccinate synthetase from the fungal pathogen<i>Cryptococcus neoformans</i>

Blundell, Ross; Williams, Simon J.; Morrow, Carl A.; Ericsson, Daniel; Kobe, Boštjan; Fraser, James S.

doi:10.1107/s1744309113021921

Cited by 3 publications

(8 citation statements)

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“…This tagged enzyme was expressed in E. coli and purified the protein by nickel affinity and size exclusion chromatography. 37 Pure recombinant C. neoformans AdSS was used to determine the steady-state parameters of the enzyme (Figure 4B). The purified enzyme was enzymatically active, as shown by an increase in adenylosuccinate-specific absorption at 280 nm.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Methods for in-frame cloning of the cDNA coding for ADE12 into expression vectors, heterologous expression in E. coli, protein purification, and crystallization of apoenzyme have been described previously. 37 In addition, purified AdSS was also cocrystallized with its substrates IMP, GTP, hadacidin, and magnesium. For cocrystallization, the protein was screened and crystallized as previously described, 37 with the following modifications: final diffraction-quality crystals were obtained using a condition from the PEG/Ion 2 crystallization screen (Hampton research) in which no crystals were detected during screening of the apoenzyme.…”

Section: ■ Discussionmentioning

confidence: 99%

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Disruption of de Novo Adenosine Triphosphate (ATP) Biosynthesis Abolishes Virulence in Cryptococcus neoformans

et al. 2016

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Opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality among immunocompromised populations worldwide. 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 adeosine triphosphate (ATP) biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. We have investigated the potential of this enzyme as an antifungal drug target, finding that loss of function results in adenine auxotrophy in C. neoformans, as well as complete loss of virulence in a murine model. Cryptococcal AdSS was expressed and purified in Escherichia coli and the enzyme's crystal structure determined, the first example of a structure of this enzyme from fungi. Together with enzyme kinetic studies, this structural information enabled comparison of the fungal enzyme with the human orthologue and revealed species-specific differences 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.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: ■ Discussionmentioning

confidence: 99%

Disruption of de Novo Adenosine Triphosphate (ATP) Biosynthesis Abolishes Virulence in Cryptococcus neoformans

et al. 2016

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“…In the adenosine monophosphate (AMP) biosynthesis branch, ADS synthetase (EC 6.3.4.4) transfers the γ-phosphate from guanosine triphosphate (GTP) to IMP, forming the intermediate 6-phosphoryl IMP; the 6-phosphoryl group is then displaced by the α-amino group of L-aspartate to form ADS. 24,25 The conversion of ADS to AMP follows the same β-elimination mechanism by ADS lyase that it performs earlier in the pathway. 26,27 AMP can be converted to adenosine diphosphateby adenylate kinase, then to ATP by nucleoside diphosphate kinase.…”

Section: The Biochemistry Of Purine Biosynthesis In the Eukaryotamentioning

confidence: 99%

Surveying purine biosynthesis across the domains of life unveils promising drug targets in pathogens

Chua

Fraser

2020

Immunol Cell Biol

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Purines play an integral role in cellular processes such as energy metabolism, cell signaling and encoding the genetic makeup of all living organisms—ensuring that the purine metabolic pathway is maintained across all domains of life. To gain a deeper understanding of purine biosynthesis via the de novo biosynthetic pathway, the genes encoding purine metabolic enzymes from 35 archaean, 69 bacterial and 99 eukaryotic species were investigated. While the classic elements of the canonical purine metabolic pathway were utilized in all domains, a subset of familiar biochemical roles was found to be performed by unrelated proteins in some members of the Archaea and Bacteria. In the Bacteria, a major differentiating feature of de novo purine biosynthesis is the increasing prevalence of gene fusions, where two or more purine biosynthesis enzymes that perform consecutive biochemical functions in the pathway are encoded by a single gene. All species in the Eukaryota exhibited the most common fusions seen in the Bacteria, in addition to new gene fusions to potentially increase metabolic flux. This complexity is taken further in humans, where a reversible biomolecular assembly of enzymes known as the purinosome has been identified, allowing short‐term regulation in response to metabolic cues while expanding on the benefits that can come from gene fusion. By surveying purine metabolism across all domains of life, we have identified important features of the purine biosynthetic pathway that can potentially be exploited as prospective drug targets.

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“…For co-crystallization, the protein was screened and crystallized as above, with the following modifications: final diffraction-quality crystals were obtained using a condition from the PEG/Ion 2 crystallization screen (Hampton research) in which no crystals were detected during screening of the apo-enzyme. Drops containing 1 µL of cocrystallization solution (AdSS protein at 16 mg/mL in gel filtration buffer from (80) , 5 mM IMP, 5 mM GTP, 5 mM hadacidin, 50 mM magnesium acetate) and 1 µL of reservoir solution (0.1 M ammonium citrate tribasic, 18% PEG3350) were equilibrated over 500 µL reservoirs. Long rectangular crystals formed at 18 ˚C after 24-48 hours, with no need for streak-seeding.…”

Section: Crystallization Of C Neoformans Adssmentioning

confidence: 99%

“…This tagged enzyme was expressed in E. coli and purified the protein by nickel affinity and sizeexclusion chromatography (80) . Pure recombinant C. neoformans AdSS was used to determine the steady-state parameters of the enzyme (Fig 9B).…”

Section: Fig 9 Enzyme Kinetics Of C Neoformans Adss (A) Adss Enzymmentioning

confidence: 99%

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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Purification, crystallization and preliminary X-ray analysis of adenylosuccinate synthetase from the fungal pathogenCryptococcus neoformans

Cited by 3 publications

References 32 publications

Disruption of de Novo Adenosine Triphosphate (ATP) Biosynthesis Abolishes Virulence in Cryptococcus neoformans

Disruption of de Novo Adenosine Triphosphate (ATP) Biosynthesis Abolishes Virulence in Cryptococcus neoformans

Surveying purine biosynthesis across the domains of life unveils promising drug targets in pathogens

Disruption of de novo ATP biosynthesis abolishes virulence in Cryptococcus neoformans

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