Adenylosuccinate synthase from <i>Saccharomyces cerevisiae</i>: homologous overexpression, purification and characterization of the recombinant protein

Lipps, Georg; Krauss, Gerhard

doi:10.1042/bj3410537

Cited by 23 publications

(12 citation statements)

References 32 publications

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“…In yeast, ADE12 encodes the adenylosuccinate synthase (AdSS) involved in the de novo synthesis of AMP from IMP (Lipps & Krauss, 1999). FgADE12 (FGSG_05187) is the ortholog of ADE12 in F. graminearum and has conserved domain sequences and active sites of AdSS (Fig.…”

Section: Resultsmentioning

confidence: 99%

Stage‐specific regulation of purine metabolism during infectious growth and sexual reproduction in Fusarium graminearum

et al. 2021

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Summary Ascospores generated during sexual reproduction are the primary inoculum for the wheat scab fungus Fusarium graminearum. Purine metabolism is known to play important roles in fungal pathogens but its lifecycle stage‐specific regulation is unclear. By characterizing the genes involved in purine de novo and salvage biosynthesis pathways, we showed that de novo syntheses of inosine, adenosine and guanosine monophosphates (IMP, AMP and GMP) are important for vegetative growth, sexual/asexual reproduction, and infectious growth, whereas purine salvage synthesis is dispensable for these stages in F. graminearum. Addition of GMP rescued the defects of the Fgimd1 mutant in vegetative growth and conidiation but not sexual reproduction, whereas addition of AMP rescued all of these defects of the Fgade12 mutant, suggesting that the function of de novo synthesis of GMP rather than AMP is distinct in sexual stages. Moreover, Acd1, an ortholog of AMP deaminase, is dispensable for growth but essential for ascosporogenesis and pathogenesis, suggesting that AMP catabolism has stage‐specific functions during sexual reproduction and infectious growth. The expression of almost all the genes involved in de novo purine synthesis is downregulated during sexual reproduction and infectious growth relative to vegetative growth. This study revealed that F. graminearum has stage‐specific regulation of purine metabolism during infectious growth and sexual reproduction.

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Section: Resultsmentioning

confidence: 99%

Stage‐specific regulation of purine metabolism during infectious growth and sexual reproduction in Fusarium graminearum

et al. 2021

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“…10 IMP•GTP•MjADSS: 6-pIMP Formation Occurs in the Absence of L-Aspartate. Extensive kinetic studies of ADSSs from various organisms such as E. coli, 32 mouse, 8 Zea mays, 33 and Saccharomyces cerevisiae 34 suggest that IMP and GTP bind in random order to the enzyme at distinct binding sites. Once both ligands are bound, structural studies show that the first catalytic intermediate, 6-pIMP, is formed even in the absence of L-aspartate in these enzymes.…”

Section: ■ Resultsmentioning

confidence: 99%

“…Extensive kinetic studies of ADSSs from various organisms such as E. coli , mouse, Zea mays , and Saccharomyces cerevisiae suggest that IMP and GTP bind in random order to the enzyme at distinct binding sites. Once both ligands are bound, structural studies show that the first catalytic intermediate, 6-pIMP, is formed even in the absence of l -aspartate in these enzymes. , By contrast, in MjADSS, it was reported that the presence of HDA was required for complete utilization of GTP, which was implicated in the formation of 6-pIMP .…”

Section: Resultsmentioning

confidence: 99%

Exquisite Modulation of the Active Site of Methanocaldococcus jannaschii Adenylosuccinate Synthetase in Forward Reaction Complexes

et al. 2016

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In enzymes that conduct complex reactions involving several substrates and chemical transformations, the active site must reorganize at each step to complement the transition state of that chemical step. Adenylosuccinate synthetase (ADSS) utilizes a molecule each of guanosine 5'-monophosphate (GTP) and aspartate to convert inosine 5'-monophosphate (IMP) into succinyl adenosine 5'-monophosphate (sAMP) through several kinetic intermediates. Here we followed catalysis by ADSS through high-resolution vibrational spectral fingerprints of each substrate and intermediate involved in the forward reaction. Vibrational spectra show differential ligand distortion at each step of catalysis, and band positions of substrates are influenced by binding of cosubstrates. We found that the bound IMP is distorted toward its N1-deprotonated form even in the absence of any other ligands. Several specific interactions between GTP and active-site amino acid residues result in large Raman shifts and contribute substantially to intrinsic binding energy. When both IMP and GTP are simultaneously bound to ADSS, IMP is converted into an intermediate 6-phosphoryl inosine 5'-monophosphate (6-pIMP). The 6-pIMP·ADSS complex was found to be stable upon binding of the third ligand, hadacidin (HDA), an analogue of l-aspartate. We find that in the absence of HDA, 6-pIMP is quickly released from ADSS, is unstable in solution, and converts back into IMP. HDA allosterically stabilizes ADSS through local conformational rearrangements. We captured this complex and determined the spectra and structure of 6-pIMP in its enzyme-bound state. These results provide important insights into the exquisite tuning of active-site interactions with changing substrate at each kinetic step of catalysis.

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“…Steady-State Enzyme Kinetics and Inhibitor Analysis. All enzyme assays were performed as previously described 38 with some modifications. Enzymatic assays were performed at 25 °C in triplicate by monitoring the production of adenylosuccinate at 280 nm (ε = 11.7 mM −1 cm −1 ) for 5 min using a Varian Cary 60 spectrophotometer (Agilent Technologies, USA).…”

Section: ■ Discussionmentioning

confidence: 99%

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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Adenylosuccinate synthase from Saccharomyces cerevisiae: homologous overexpression, purification and characterization of the recombinant protein

Cited by 23 publications

References 32 publications

Stage‐specific regulation of purine metabolism during infectious growth and sexual reproduction in Fusarium graminearum

Stage‐specific regulation of purine metabolism during infectious growth and sexual reproduction in Fusarium graminearum

Exquisite Modulation of the Active Site of Methanocaldococcus jannaschii Adenylosuccinate Synthetase in Forward Reaction Complexes

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

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