Purine metabolism in plant pathogenic fungi.

In eukaryotic cells, purine metabolism is the way to the production of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and plays key roles in various biological processes. Purine metabolism mainly consists of de novo, salvage, and catabolic pathways, and some components of these pathways have been characterized in some plant pathogenic fungi, such as the rice blast fungus Magnaporthe oryzae and wheat head blight fungus Fusarium graminearum. The enzymatic steps of the de novo pathway are well-conserved in plant pathogenic fungi and play crucial roles in fungal growth and development. Blocking this pathway inhibits the formation of penetration structures and invasive growth, making it essential for plant infection by pathogenic fungi. The salvage pathway is likely indispensable but requires exogenous purines, implying that purine transporters are functional in these fungi. The catabolic pathway balances purine nucleotides and may have a conserved stage-specific role in pathogenic fungi. The significant difference of the catabolic pathway in planta and in vitro lead us to further explore and identify the key genes specifically regulating pathogenicity in purine metabolic pathway. In this review, we summarized recent advances in the studies of purine metabolism, focusing on the regulation of pathogenesis and growth in plant pathogenic fungi.

AICAR transformylase/IMP cyclohydrolase (ATIC) is essential for de novo purine biosynthesis and infection by Cryptococcus neoformans.

The fungal pathogen Cryptococcus neoformans is a leading cause of meningoencephalitis in the immunocompromised. As current antifungal treatments are toxic to the host, costly, limited in their efficacy, and associated with drug resistance, there is an urgent need to identify vulnerabilities in fungal physiology to accelerate antifungal discovery efforts. Rational drug design was pioneered in de novo purine biosynthesis as the end products of the pathway, ATP and GTP, are essential for replication, transcription, and energy metabolism, and the same rationale applies when considering the pathway as an antifungal target. Here, we describe the identification and characterization of C. neoformans 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/5'-inosine monophosphate cyclohydrolase (ATIC), a bifunctional enzyme that catalyzes the final two enzymatic steps in the formation of the first purine base inosine monophosphate. We demonstrate that mutants lacking the ATIC-encoding ADE16 gene are adenine and histidine auxotrophs that are unable to establish an infection in a murine model of virulence. In addition, our assays employing recombinantly expressed and purified C. neoformans ATIC enzyme revealed Km values for its substrates AICAR and 5-formyl-AICAR are 8-fold and 20-fold higher, respectively, than in the human ortholog. Subsequently, we performed crystallographic studies that enabled the determination of the first fungal ATIC protein structure, revealing a key serine-to-tyrosine substitution in the active site, which has the potential to assist the design of fungus-specific inhibitors. Overall, our results validate ATIC as a promising antifungal drug target.

Combined Targeted and Untargeted Profiling of HeLa Cells Deficient in Purine De Novo Synthesis.

Three genetically determined enzyme defects of purine de novo synthesis (PDNS) have been identified so far in humans: adenylosuccinate lyase (ADSL) deficiency, 5-amino-4-imidazole carboxamide-ribosiduria (AICA-ribosiduria), and deficiency in bifunctional enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS). Clinical signs of these defects are mainly neurological, such as seizures, psychomotor retardation, epilepsy, autistic features, etc. This work aims to describe the metabolic changes of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual steps of PDNS to better understand known and potential defects of the pathway in humans. High-performance liquid chromatography coupled with mass spectrometry was used for both targeted and untargeted metabolomic analyses. The statistically significant features from the untargeted study were identified by fragmentation analysis. Data from the targeted analysis were processed in Cytoscape software to visualize the most affected metabolic pathways. Statistical significance of PDNS intermediates preceding deficient enzymes was the highest (p-values 10 × 10-7-10 × 10-15) in comparison with the metabolites from other pathways (p-values of up to 10 × 10-7). Disturbed PDNS resulted in an altered pool of adenine and guanine nucleotides. However, the adenylate energy charge was not different from controls. Different profiles of acylcarnitines observed among deficient cell lines might be associated with a specific enzyme deficiency rather than global changes related to the PDNS pathway. Changes detected in one-carbon metabolism might reduce the methylation activity of the deficient cells, thus affecting the modification state of DNA, RNA, and proteins.

Disorders of purine biosynthesis metabolism.

Purines are essential molecules that are components of vital biomolecules, such as nucleic acids, coenzymes, signaling molecules, as well as energy transfer molecules. The de novo biosynthesis pathway starts from phosphoribosylpyrophosphate (PRPP) and eventually leads to the synthesis of inosine monophosphate (IMP) by means of 10 sequential steps catalyzed by six different enzymes, three of which are bi-or tri-functional in nature. IMP is then converted into guanosine monophosphate (GMP) or adenosine monophosphate (AMP), which are further phosphorylated into nucleoside di- or tri-phosphates, such as GDP, GTP, ADP and ATP. This review provides an overview of inborn errors of metabolism pertaining to purine synthesis in humans, including either phosphoribosylpyrophosphate synthetase (PRS) overactivity or deficiency, as well as adenylosuccinate lyase (ADSL), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS), and adenylosuccinate synthetase (ADSS) deficiencies. ITPase deficiency is being described as well. The clinical spectrum of these disorders is broad, including neurological impairment, such as psychomotor retardation, epilepsy, hypotonia, or microcephaly; sensory involvement, such as deafness and visual disturbances; multiple malformations, as well as muscle presentations or consequences of hyperuricemia, such as gouty arthritis or kidney stones. Clinical signs are often nonspecific and, thus, overlooked. It is to be hoped that this is likely to be gradually overcome by using sensitive biochemical investigations and next-generation sequencing technologies.

Dietary intake of fructose increases purine de novo synthesis: A crucial mechanism for hyperuricemia.

Background: Fructose consumption is a potential risk factor for hyperuricemia because uric acid (UA) is a byproduct of fructose metabolism caused by the rapid consumption of adenosine triphosphate and accumulation of adenosine monophosphate (AMP) and other purine nucleotides. Additionally, a clinical experiment with four gout patients demonstrated that intravenous infusion of fructose increased the purine de novo synthesis rate, which implied fructose-induced hyperuricemia might be related to purine nucleotide synthesis. Moreover, the mechanistic (mammalian) target of rapamycin (mTOR) is a key protein both involved in fructose metabolism and purine de novo synthesis. The present study was conducted to elucidate how fructose influences mTOR and purine de novo synthesis in a hepatic cell line and livers of mice. Materials and methods: RNA-sequencing in NCTC 1469 cells treated with 0- and 25-mM fructose for 24 h and metabolomics analysis on the livers of mice fed with 0- and 30-g/kg fructose for 2 weeks were assessed. Gene and protein expression of phosphoribosyl pyrophosphate synthase (PRPSAP1), Glutamine PRPP aminotransferase (PPAT), adenyl succinate lyase (ADSL), adenyl succinate synthetase isozyme-1 (Adss1), inosine-5'-monophosphate dehydrogenase (IMPDH), and guanine monophosphate synthetase (GMPS) was measured. The location of PRPSAP1 and PPAT in the liver was assessed by an immunofluorescence assay. Results: Metabolite profiling showed that the level of AMP, adenine, adenosine, hypoxanthine, and guanine was increased significantly. RNA-sequencing showed that gene expression of phosphoribosyl pyrophosphate synthase (PRPS2), phosphoribosyl glycinamide formyl transferase (GART), AICAR transformylase (ATIC), ADSL, Adss1, and IMPDH were raised, and gene expression of adenosine monophosphate deaminase 3 (AMPD3), adenosine deaminase (ADA), 5',3'-nucleotidase, cytosolic (NT5C), and xanthine oxidoreductase (XOR) was also increased significantly. Fructose increased the gene expression, protein expression, and fluorescence intensity of PRPSAP1 and PPAT in mice livers by increasing mTOR expression. Fructose increased the expression and activity of XOR, decreased the expression of uricase, and increased the serum level of UA. Conclusion: This study demonstrated that the increased purine de novo synthesis may be a crucial mechanism for fructose-induced hyperuricemia.

Structural features of Cryptococcus neoformans bifunctional GAR/AIR synthetase may present novel antifungal drug targets.

Cryptococcus neoformans is a fungus that causes life-threatening systemic mycoses. During infection of the human host, this pathogen experiences a major change in the availability of purines; the fungus can scavenge the abundant purines in its environmental niche of pigeon excrement, but must employ de novo biosynthesis in the purine-poor human CNS. Eleven sequential enzymatic steps are required to form the first purine base, IMP, an intermediate in the formation of ATP and GTP. Over the course of evolution, several gene fusion events led to the formation of multifunctional purine biosynthetic enzymes in most organisms, particularly the higher eukaryotes. In C. neoformans, phosphoribosyl-glycinamide synthetase (GARs) and phosphoribosyl-aminoimidazole synthetase (AIRs) are fused into a bifunctional enzyme, while the human ortholog is a trifunctional enzyme that also includes GAR transformylase. Here we functionally, biochemically, and structurally characterized C. neoformans GARs and AIRs to identify drug targetable features. GARs/AIRs are essential for de novo purine production and virulence in a murine inhalation infection model. Characterization of GARs enzymatic functional parameters showed that C. neoformans GARs/AIRs have lower affinity for substrates glycine and PRA compared with the trifunctional metazoan enzyme. The crystal structure of C. neoformans GARs revealed differences in the glycine- and ATP-binding sites compared with the Homo sapiens enzyme, while the crystal structure of AIRs shows high structural similarity compared with its H. sapiens ortholog as a monomer but differences as a dimer. The alterations in functional and structural characteristics between fungal and human enzymes could potentially be exploited for antifungal development.

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

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.

Interplay between adenylate metabolizing enzymes and AMP-activated protein kinase.

Purine nucleotides are involved in a variety of cellular functions, such as energy storage and transfer, and signalling, in addition to being the precursors of nucleic acids and cofactors of many biochemical reactions. They can be generated through two separate pathways, the de novo biosynthesis pathway and the salvage pathway. De novo purine biosynthesis leads to the formation of IMP, from which the adenylate and guanylate pools are generated by two additional steps. The salvage pathways utilize hypoxanthine, guanine and adenine to generate the corresponding mononucleotides. Despite several decades of research on the subject, new and surprising findings on purine metabolism are constantly being reported, and some aspects still need to be elucidated. Recently, purine biosynthesis has been linked to the metabolic pathways regulated by AMP-activated protein kinase (AMPK). AMPK is the master regulator of cellular energy homeostasis, and its activity depends on the AMP : ATP ratio. The cellular energy status and AMPK activation are connected by AMP, an allosteric activator of AMPK. Hence, an indirect strategy to affect AMPK activity would be to target the pathways that generate AMP in the cell. Herein, we report an up-to-date review of the interplay between AMPK and adenylate metabolizing enzymes. Some aspects of inborn errors of purine metabolism are also discussed.

Catalytic pathway, substrate binding and stability in SAICAR synthetase: A structure and molecular dynamics study.

The de novo purine biosynthesis is one of the highly conserved pathways among all organisms and is essential for the cell viability. A clear understanding of the enzymes in this pathway would pave way for the development of antimicrobial and anticancer drugs. Phosphoribosylaminoimidazole-succinocarboxamide (SAICAR) synthetase is one of the enzymes in this pathway that catalyzes ATP dependent ligation of carboxyaminoimidazole ribotide (CAIR) with l-aspartate (ASP). Here, we describe eight crystal structures of this enzyme, in C2221 and H3 space groups, bound to various substrates and substrate mimics from a hyperthermophilic archaea Pyrococcus horikoshii along with molecular dynamics simulations of the structures with substrates. Complexes exhibit minimal deviation from its apo structure. The CAIR binding site displays a preference for pyrimidine nucleotides. In the ADP·TMP·ASP complex, the ASP binds at a position equivalent to that found in Saccharomyces cerevisiae structure (PDB: 2CNU) and thus, clears the ambiguity regarding ASP's position. A possible mode for the inhibition of the enzyme by CTP and UTP, observed earlier in the yeast enzyme, is clearly illustrated in the structures bound to CMP and UMP. The ADP.Mg(2+)·PO4·CD/MP complex having a phosphate ion between the ATP and CAIR sites strengthens one of the two probable pathways (proposed in Escherichia coli study) of catalytic mechanism and suggests the possibility of a phosphorylation taking place before the ASP's attack on CAIR. Molecular dynamic simulations of this enzyme along with its substrates at 90°C reveal the relative strengths of substrate binding, possible antagonism and the role of Mg(2+) ions.