Purine Biosynthesis Enzyme Research Articles

Targeting LRRK2 mRNA stability in Parkinson’s disease

In a recent study, Liu and colleagues demonstrated a role for the purine biosynthesis enzyme ATIC and its substrate in regulating the protein levels of the Parkinson’s disease kinase LRRK2, which rescues neurodegeneration and neuroinflammation in distinct animal models. This work highlights a novel avenue to target LRRK2 protein levels as a strategy to prevent neurodegeneration in Parkinson’s disease.

Klebsiella pneumoniae causes bacteremia using factors that mediate tissue-specific fitness and resistance to oxidative stress.

Gram-negative bacteremia is a major cause of global morbidity involving three phases of pathogenesis: initial site infection, dissemination, and survival in the blood and filtering organs. Klebsiella pneumoniae is a leading cause of bacteremia and pneumonia is often the initial infection. In the lung, K. pneumoniae relies on many factors like capsular polysaccharide and branched chain amino acid biosynthesis for virulence and fitness. However, mechanisms directly enabling bloodstream fitness are unclear. Here, we performed transposon insertion sequencing (TnSeq) in a tail-vein injection model of bacteremia and identified 58 K. pneumoniae bloodstream fitness genes. These factors are diverse and represent a variety of cellular processes. In vivo validation revealed tissue-specific mechanisms by which distinct factors support bacteremia. ArnD, involved in Lipid A modification, was required across blood filtering organs and supported resistance to soluble splenic factors. The purine biosynthesis enzyme PurD supported liver fitness in vivo and was required for replication in serum. PdxA, a member of the endogenous vitamin B6 biosynthesis pathway, optimized replication in serum and lung fitness. The stringent response regulator SspA was required for splenic fitness yet was dispensable in the liver. In a bacteremic pneumonia model that incorporates initial site infection and dissemination, splenic fitness defects were enhanced. ArnD, PurD, DsbA, SspA, and PdxA increased fitness across bacteremia phases and each demonstrated unique fitness dynamics within compartments in this model. SspA and PdxA enhanced K. pnuemoniae resistance to oxidative stress. SspA, but not PdxA, specifically resists oxidative stress produced by NADPH oxidase Nox2 in the lung, spleen, and liver, as it was a fitness factor in wild-type but not Nox2-deficient (Cybb-/-) mice. These results identify site-specific fitness factors that act during the progression of Gram-negative bacteremia. Defining K. pneumoniae fitness strategies across bacteremia phases could illuminate therapeutic targets that prevent infection and sepsis.

NSUN2-mediated m5 C RNA methylation dictates retinoblastoma progression through promoting PFAS mRNA stability and expression.

The precise temporal and spatial regulation of N5 -methylcytosine (m5 C) RNA modification plays essential roles in RNA metabolism, and is necessary for the maintenance of epigenome homeostasis. Howbeit, the mechanism underlying the m5 C modification in carcinogenesis remains to be fully addressed. Global and mRNA m5 C levels were determined by mRNA isolation and anti-m5 C dot blot in both retinoblastoma (RB) cells and clinical samples. Orthotopic intraocular xenografts were established to examine the oncogenic behaviours of RB. Genome-wide multiomics analyses were performed to identify the functional target of NSUN2, including proteomic analysis, transcriptome screening and m5 C-methylated RNA immunoprecipitation sequencing (m5 C-meRIP-seq). Organoid-based single-cell analysis and gene-correlation analysis were performed to verify the NSUN2/ALYREF/m5 C-PFAS oncogenic cascade. Herein, we report that NSUN2-mediated m5 C RNA methylation fuels purine biosynthesis during the oncogenic progression of RB. First, we discovered that global and mRNA m5 C levels were significantly enriched in RBs compared to normal retinas. In addition, tumour-specific NSUN2 expression was noted in RB samples and cell lines. Therapeutically, targeted correction of NSUN2 exhibited efficient therapeutic efficacy in RB both in vitro and in vivo. Through multiomics analyses, we subsequently identified phosphoribosylformylglycinamidine synthase (PFAS), a vital enzyme in purine biosynthesis, as a downstream candidate target of NSUN2. The reintroduction of PFAS largely reversed the inhibitory phenotypes in NSUN2-deficient RB cells, indicating that PFAS was a functional downstream target of NSUN2. Mechanistically, we found that the m5 C reader protein ALYREF was responsible for the recognition of the m5 C modification of PFAS, increasing its expression by enhancing its RNA stability. Conclusively, we initially demonstrated that NSUN2 is necessary for oncogenic gene activation in RB, expanding the current understanding of dynamic m5 C function during tumour progression. As the NSUN2/ALYREF/m5 C-PFAS oncogenic cascade is an important RB trigger, our study suggests that a targeted m5 C reprogramming therapeutic strategy may be a novel and efficient anti-tumour therapy approach.

Structural basis of the substrate recognition and inhibition mechanism of Plasmodium falciparum nucleoside transporter PfENT1

By lacking de novo purine biosynthesis enzymes, Plasmodium falciparum requires purine nucleoside uptake from host cells. The indispensable nucleoside transporter ENT1 of P. falciparum facilitates nucleoside uptake in the asexual blood stage. Specific inhibitors of PfENT1 prevent the proliferation of P. falciparum at submicromolar concentrations. However, the substrate recognition and inhibitory mechanism of PfENT1 are still elusive. Here, we report cryo-EM structures of PfENT1 in apo, inosine-bound, and inhibitor-bound states. Together with in vitro binding and uptake assays, we identify that inosine is the primary substrate of PfENT1 and that the inosine-binding site is located in the central cavity of PfENT1. The endofacial inhibitor GSK4 occupies the orthosteric site of PfENT1 and explores the allosteric site to block the conformational change of PfENT1. Furthermore, we propose a general “rocker switch” alternating access cycle for ENT transporters. Understanding the substrate recognition and inhibitory mechanisms of PfENT1 will greatly facilitate future efforts in the rational design of antimalarial drugs.

Abstract 2215: Human metapneumovirus recruits purine biosynthesis enzymes to phase-separated compartments to regulate viral replication

Abstract 2215: Human metapneumovirus recruits purine biosynthesis enzymes to phase-separated compartments to regulate viral replication

Identification of genetic elements required for Listeria monocytogenes growth under limited nutrient conditions and virulence by a screening of transposon insertion library.

Listeria monocytogenes, the causative agent of listeriosis, displays a lifestyle ranging from saprophytes in the soil to pathogenic as a facultative intracellular parasite in host cells. In the current study, a random transposon (Tn) insertion library was constructed in L. monocytogenes strain F2365 and screened to identify genes and pathways affecting in vitro growth and fitness in minimal medium (MM) containing different single carbohydrate as the sole carbon source. About 2,000 Tn-mutants were screened for impaired growth in MM with one of the following carbon sources: glucose, fructose, mannose, mannitol, sucrose, glycerol, and glucose 6-phosphate (G6P). Impaired or abolished growth of L. monocytogenes was observed for twenty-one Tn-mutants with disruptions in genes encoding purine biosynthesis enzymes (purL, purC, purA, and purM), pyrimidine biosynthesis proteins (pyrE and pyrC), ATP synthase (atpI and atpD2), branched-chain fatty acids (BCFA) synthesis enzyme (bkdA1), a putative lipoprotein (LMOF2365_2387 described as LP2387), dUTPase family protein (dUTPase), and two hypothetical proteins. All Tn-mutants, except the atpD2 mutant, grew as efficiently as wild-type strain in a nutrient rich media. The virulence of twenty-one Tn-mutants was assessed in mice at 72 h following intravenous (IV) infection. The most attenuated mutants had Tn insertions in purA, hypothetical protein (LMOf2365_0064 described as HP64), bkdA1, dUTPase, LP2387, and atpD2, confirming the important role of these genes in pathogenesis. Six Tn-mutants were then tested for ability to replicate intracellularly in murine macrophage J774.1 cells. Significant intracellular growth defects were observed in two Tn-mutants with insertions in purA and HP64 genes, suggesting that an intact purine biosynthesis pathway is important for intracellular growth of L. monocytogens. These findings may not be fully generalized to all of L. monocytogenes strains due to their genetic diversity. In conclusion, Tn-mutagenesis identified that biosynthesis of purines, pyrimidines, ATP, and BCFA are important for L. monocytogens pathogenesis. Purine and pyrimidine auxotrophs play an important role in the pathogenicity in other bacterial pathogens, but our study also revealed new proteins essential for both growth in MM and L. monocytogenes strain F2365 virulence.

Purine biosynthesis and related enzymes play the oncogenic function in FGFR3 mutant urothelial carcinoma

Urothelial carcinoma (UC), as one of the malignant tumors, has a high incidence, mortality, and poor survival rate. FGFR3 is one of the frequency mutation genetic events of UC. Recent studies have reported that FGFR3 mutations are associated with lower aggressiveness and better prognosis of UC, and small molecule compounds with FGFR3 mutations have been developed and treated. However, they are still limited, especially for wild‐type cells. We used Ingenuity Pathways Analysis (IPA) to analyze the significantly expression of 351 genes (>2‐fold change cutoff value) between FGFR3 wild‐type and mutation/fusion UC cells by FGFR3 inhibitors treatment. Through the predicted canonical pathways, several metabolism‐related events have been regulated, including purine biosynthesis. Focusing on de novo purine biosynthesis, we found that several involved enzymes are upregulated in UC. In addition, we have also observed that several enzymes are inhibited by FGFR3 inhibitors in mutant cells, but not in wild type. We then hypothesized that purine biosynthetic events might confer drug resistance and promote UC tumorigenicity. To verify our observations, we determined the expression level of purine biosynthesis after FGFR3 classification. The results indicate a similar trend in clinical cohorts that overexpression of purine biosynthesis genes in FGFR3 wild‐type. Moreover, combining these genes can be used as an independent prognostic factor (HR=1.41, p=0.026). We further established the overexpression of purine biosynthesis genes in some FGFR3 mutant UC cells. We determined that purine biosynthesis genes cause FGFR3 mutations models to be more prone to mesenchymal and metastatic than controls. Our studies also observed that FGFR3 wild‐type cells promote UC cell radioresistance via purine biosynthesis and further activate the homologous recombination repair mechanism. Combining all evidence, we claim that purine biosynthesis should be focused and regarded as a novel strategy against UC.

A Review of Small-Molecule Inhibitors of One-Carbon Enzymes: SHMT2 and MTHFD2 in the Spotlight.

Metabolic reprogramming is a key hallmark of cancer and shifts cellular metabolism to meet the demands of biomass production necessary for abnormal cell reproduction. One-carbon metabolism (1CM) contributes to many biosynthetic pathways that fuel growth and is comprised of a complex network of enzymes. Methotrexate and 5-fluorouracil were pioneering drugs in this field and are still widely used today as anticancer agents as well as for other diseases such as arthritis. Besides dihydrofolate reductase and thymidylate synthase, two other enzymes of the folate cycle arm of 1CM have not been targeted clinically: serine hydroxymethyltransferase (SHMT) and methylenetetrahydrofolate dehydrogenase (MTHFD). An increasing body of literature suggests that the mitochondrial isoforms of these enzymes (SHMT2 and MTHFD2) are clinically relevant in the context of cancer. In this review, we focused on the 1CM pathway as a target for cancer therapy and, in particular, SHMT2 and MTHFD2. The function, regulation, and clinical relevance of SHMT2 and MTHFD2 are all discussed. We expand on previous clinical studies and evaluate the prognostic significance of these critical enzymes by performing a pan-cancer analysis of patient data from the The Cancer Genome Atlas and a transcriptional coexpression network enrichment analysis. We also provide an overview of preclinical and clinical inhibitors targeting the folate pathway, the methionine cycle, and folate-dependent purine biosynthesis enzymes.

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

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.

Abstract 4800: Targeting mitochondrial and cytosolic one-carbon metabolism in epithelial ovarian cancer via folate receptor alpha

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Even though most patients initially respond to platinum-based therapy, the likelihood of disease reoccurrence is virtually 100%. Thus, there is an urgent need for new tumor-selective therapies for EOC. One such treatment option involves targeting tumors via folate receptor alpha (FRα) which is overexpressed in up to 90% of EOCs and shows increasing expression with stage and grade of disease. Our laboratory discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine analogs (AGF291, AGF320, AGF347, AGF359 and AGF362) which inhibit mitochondrial one-carbon (C1) metabolism at serine hydroxymethyltransferase (SHMT) 2, with secondary inhibitions at cytosolic enzyme targets including those in de novo purine biosynthesis and SHMT1. Potent inhibition was seen toward isogenic Chinese hamster ovary (CHO) cell lines individually expressing FRα, the reduced folate carrier (RFC, ubiquitously expressed tissue folate transporter) and the proton-coupled folate transporter (PCFT, expressed in a limited number of normal tissues and inactive at normal pH, and several solid tumors at acidic pH including EOC), and with FRα-expressing tumor cells, including KB and IGROV1, a human EOC cell line. Inhibitory potencies were in order, AGF347 > AGF362 >> AGF291 = AGF320 = AGF359. Drug effects were substantially reduced with excess folic acid, confirming FRα-mediated drug uptake. Toward cisplatin resistant SKOV3, TOV112D and A2780 EOC cells, inhibition in the nanomolar range was detected with all compounds. Excess folic acid abrogated drug effects to varying degrees, suggesting significant uptake by the PCFT and/or the RFC, in addition to FRα. Short-term (5 minutes) cell uptake assays with the EOC cell lines and [3H]AGF347 confirmed transport by PCFT and RFC. With sustained exposures resulting in steady state AGF347 accumulations under both physiologic (pH 7.2) and acidic (pH 6.8, approximating the tumor microenvironment) conditions, FRα uptake predominated. [3H]AGF347 treatment of IGROV1 EOC resulted in substantial drug accumulation in both cytosol and mitochondria. Inhibition of cell proliferation for all analogs was reversed by addition of both glycine and adenosine, implicating C1 metabolism in mitochondria including SHMT2 and de novo purine biosynthesis in the cytosol as the targeted pathways; protection by 5-aminoimidazole-4-carboxamide (AICA) with or without glycine was incomplete, implying direct targeting of AICA ribonucleotide formyltransferase (AICARFTase), the second folate-dependent enzyme in purine biosynthesis. Our studies describe first-in-class FRα-selective compounds targeting mitochondrial and cytosolic C1 metabolism, with potent activity against EOC, including cisplatin resistant EOC. Citation Format: Adrianne Wallace-Povirk, Carrie O’Connor, Aamod Dekhne, Zhanjun Hou, Md. Junayed Nayeen, Khushbu Shah, Aleem Gangjee, Larry Matherly. Targeting mitochondrial and cytosolic one-carbon metabolism in epithelial ovarian cancer via folate receptor alpha [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4800.

Increase of PRPP enhances chemosensitivity of PRPS1 mutant acute lymphoblastic leukemia cells to 5‐Fluorouracil

Relapse‐specific mutations in phosphoribosyl pyrophosphate synthetase 1 (PRPS1), a rate‐limiting purine biosynthesis enzyme, confer significant drug resistances to combination chemotherapy in acute lymphoblastic leukemia (ALL). It is of particular interest to identify drugs to overcome these resistances. In this study, we found that PRPS1 mutant ALL cells specifically showed more chemosensitivity to 5‐Fluorouracil (5‐FU) than control cells, attributed to increased apoptosis of PRPS1 mutant cells by 5‐FU. Mechanistically, PRPS1 mutants increase the level of intracellular phosphoribosyl pyrophosphate (PRPP), which causes the apt conversion of 5‐FU to FUMP and FUTP in Reh cells, to promote 5‐FU‐induced DNA damage and apoptosis. Our study not only provides mechanistic rationale for re‐targeting drug resistant cells in ALL, but also implicates that ALL patients who harbor relapse‐specific mutations of PRPS1 might benefit from 5‐FU‐based chemotherapy in clinical settings.

Purine Biosynthesis Enzymes in Hippocampal Neurons.

Despite reports implicating disrupted purine metabolism in causing a wide spectrum of neurological defects, the mechanistic details of purine biosynthesis in neurons are largely unknown. As an initial step in filling that gap, we examined the expression and subcellular distribution of three purine biosynthesis enzymes (PFAS, PAICS and ATIC) in rat hippocampal neurons. Using immunoblotting and high-resolution light and electron microscopic analysis, we find that all three enzymes are broadly distributed in hippocampal neurons with pools of these enzymes associated with mitochondria. These findings suggest a potential link between purine metabolism and mitochondrial function in neurons and provide an impetus for further studies.

De Novo Purine Biosynthesis in Drug Resistance and Tumor Relapse of Childhood ALL

Background: Relapse is the leading cause of mortality in children with acute lymphoblastic leukemia (ALL). Studies have shown that most ALL cases are polyclonal at diagnosis and that genetic changes in individual subclones influence sensitity to therapy and subsequent clonal evolution during therapy; but the molecular details remain to be worked out. Among different pathways enriched for mutations at relapse, purine metabolism is particularly interesting for two reasons: first, thiopurines are widely used in the ALL combination chemotherapy regimens, and are prodrugs that are converted by the purine salvage pathway to cytotoxic metabolites. Second, de novo nucleotide biosynthesis is often upregulated in cancer cells, and it is believed that sufficient nucleotide pools are required to maintain genomic stability, could bypass oncogene-induced senescence and promote tumor progression1. Therefore, we focus our current study on de novo purine biosynthesis in drug resistance and tumor relapse of childhood ALL.Methods and Results: Using whole-exome sequencing, we identified relapse-specific mutations in the phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1), which encodes a rate-limiting purine biosynthesis enzyme, in 24/358 (6.7%) relapsed childhood B cell ALL (B-ALL) cases. Targeted sequencing identified mutations in additional genes in de novo purine biosynthesis pathway, providing further genetic evidence for its importance in relapsed ALL. All individuals with PRPS1 mutation relapsed early on-treatment (P <0.001), having an inferior prognosis1.Using various functional assays, we demonstrated that rather than causing a simple gain-of-function effect, the mutations in PRPS1 resulted in the disruption of the normal feedback inhibition of purine synthesis, in which the enzyme remained active despite an increased concentration of nucleoside analogs. PRPS1 mutants increased synthesis of the nucleoside inosine monophosphate, its metabolite hypoxanthine (HX) and de novo purine biosynthesis intermediates (e.g. AICAR, SAICAR) in Reh cells. Increased intracellular HX can competively inhibit the conversion of thiopurines into their active metabolites. Furthermore, inhibition of de novo purine biosynthesis in vitro, either by CRISPR-Cas9 genome editing of de novo purine synthesis pathway genes (GART, ATIC etc.) or treatment with a pathway inhibitor lometrexol (GART inhibitor) alleviated the metabolic disturbance and drug resistance induced by PRPS1 mutations.Using ultra-deep sequencing of unique serial remission samples before clinical relapse, we noticed that the PRPS1 mutant allele fraction increased drastically before clinical relapse, suggesting rapid clonal expansion occurs after the acquisition of a PRPS1 mutation. Interestingly, we also noticed that PPRS1 mutation coexist with RAS mutation in many relapse cases and at single cell resolution. Functional analysis revealed that tumor cells which harbored RAS and PRPS1 double mutations are more drug resistant than those with RAS or PRPS1 mutation alone. Previous studies have shown that oncogenic RAS mutation can also induce various stress responses including oncogene-induced senensence and DNA damage response (DDR), which all could impede tumor cell proliferation during relapse. In vitro, we found PRPS1 mutation can release the replication and metabolic stress caused by RAS mutation, in addition to their role in thiopurine resistance. The PRPS1 mutants not only increase the nucleotide pools but also elevate purine biosynthesis intermediate AICAR, which can activate AMPK and reduce the RAS mutant-induced DDR. We are currently working on in vitro and in vivo models (including patient derived xenograft models) to further test the double mutant's effects on tumor-reinitiation and clonal evolution during ALL relapse.Conclusions: We demonstrated that negative feedback-defective PRPS1 mutants can drive de novo purine biosynthesis, which can exert drug resistance and reduce genomic instability during tumor relapse. Our study highlights the importance of de novo purine biosynthesis in the pathogenesis of relapse, and suggests a diagnostic approach to predicting early relapse and a therapeutic strategy to circumventing resistance in ALL.1 Li et al. Negative feedback-defective PRPS1 mutants drivee thiopurine resistance in relapsed childhood ALL. Nature Medicine,21(6): 563-571 (2015) DisclosuresNo relevant conflicts of interest to declare.

Thiopurine Resistance in Childhood ALL Is Mediated by PRPS1 Mutations.

Mutations in the de novo purine biosynthesis enzyme PRPS1 drive thiopurine resistance in relapsed ALL.

Negative feedback-defective PRPS1 mutants drive thiopurine resistance in relapsed childhood ALL.

Relapse is the leading cause of mortality in children with acute lymphoblastic leukemia (ALL). Among chemotherapeutics, thiopurines are key drugs in ALL combination therapy. Using whole-exome sequencing, we identified relapse-specific mutations in the phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1), which encodes a rate-limiting purine biosynthesis enzyme, in 24/358 (6.7%) relapsed childhood B cell ALL (B-ALL) cases. All individuals who harbored PRPS1 mutations relapsed early during treatment, and mutated ALL clones expanded exponentially before clinical relapse. Our functional analyses of PRPS1 mutants uncovered a new chemotherapy-resistance mechanism involving reduced feedback inhibition of de novo purine biosynthesis and competitive inhibition of thiopurine activation. Notably, the de novo purine synthesis inhibitor lometrexol effectively abrogated PRPS1 mutant-driven drug resistance. These results highlight the importance of constitutive activation of the de novo purine synthesis pathway in thiopurine resistance, and they offer therapeutic strategies for the treatment of relapsed and thiopurine-resistant ALL.

Metal stopping reagents facilitate discontinuous activity assays of the de novo purine biosynthesis enzyme PurE

The conversion of 5-aminoimidazole ribonucleotide (AIR) to 4-carboxy-AIR (CAIR) represents an unusual divergence in purine biosynthesis: microbes and nonmetazoan eukaryotes use class I PurEs while animals use class II PurEs. Class I PurEs are therefore a potential antimicrobial target; however, no enzyme activity assay is suitable for high throughput screening (HTS). Here we report a simple chemical quench that fixes the PurE substrate/product ratio for 24h, as assessed by the Bratton–Marshall assay (BMA) for diazotizable amines. The ZnSO4 stopping reagent is proposed to chelate CAIR, enabling delayed analysis of this acid-labile product by BMA or other HTS methods.

Revisiting and revising the purinosome

Some metabolic pathway enzymes are known to organize into multi-enzyme complexes for reasons of catalytic efficiency, metabolite channeling, and other advantages of compartmentalization. It has long been an appealing prospect that de novo purine biosynthesis enzymes form such a complex, termed the "purinosome." Early work characterizing these enzymes garnered scarce but encouraging evidence for its existence. Recent investigations led to the discovery in human cell lines of purinosome bodies-cytoplasmic puncta containing transfected purine biosynthesis enzymes, which were argued to correspond to purinosomes. New discoveries challenge both the functional and physiological relevance of these bodies in favor of protein aggregation.

Crystal structure of the invertebrate bifunctional purine biosynthesis enzyme PAICS at 2.8 Å resolution

Two important steps of the de novo purine biosynthesis pathway are catalyzed by the 5-aminoimidazole ribonucleotide carboxylase and the 4-(N-succinylcarboxamide)-5-aminoimidazole ribonucleotide synthetase enzymes. In most eukaryotic organisms, these two activities are present in the bifunctional enzyme complex known as PAICS. We have determined the 2.8-Å resolution crystal structure of the 350-kDa invertebrate PAICS from insect cells (Trichoplusia ni) using single-wavelength anomalous dispersion methods. Comparison of insect PAICS to human and prokaryotic homologs provides insights into substrate binding and reveals a highly conserved enzymatic framework across divergent species.

Transiently Transfected Purine Biosynthetic Enzymes Form Stress Bodies

It has been hypothesized that components of enzymatic pathways might organize into intracellular assemblies to improve their catalytic efficiency or lead to coordinate regulation. Accordingly, de novo purine biosynthesis enzymes may form a purinosome in the absence of purines, and a punctate intracellular body has been identified as the purinosome. We investigated the mechanism by which human de novo purine biosynthetic enzymes might be organized into purinosomes, especially under differing cellular conditions. Irregardless of the activity of bodies formed by endogenous enzymes, we demonstrate that intracellular bodies formed by transiently transfected, fluorescently tagged human purine biosynthesis proteins are best explained as protein aggregation.

Purine biosynthesis in archaea: variations on a theme

BackgroundThe ability to perform de novo biosynthesis of purines is present in organisms in all three domains of life, reflecting the essentiality of these molecules to life. Although the pathway is quite similar in eukaryotes and bacteria, the archaeal pathway is more variable. A careful manual curation of genes in this pathway demonstrates the value of manual curation in archaea, even in pathways that have been well-studied in other domains.ResultsWe searched the Integrated Microbial Genome system (IMG) for the 17 distinct genes involved in the 11 steps of de novo purine biosynthesis in 65 sequenced archaea, finding 738 predicted proteins with sequence similarity to known purine biosynthesis enzymes. Each sequence was manually inspected for the presence of active site residues and other residues known or suspected to be required for function.Many apparently purine-biosynthesizing archaea lack evidence for a single enzyme, either glycinamide ribonucleotide formyltransferase or inosine monophosphate cyclohydrolase, suggesting that there are at least two more gene variants in the purine biosynthetic pathway to discover. Variations in domain arrangement of formylglycinamidine ribonucleotide synthetase and substantial problems in aminoimidazole carboxamide ribonucleotide formyltransferase and inosine monophosphate cyclohydrolase assignments were also identified.Manual curation revealed some overly specific annotations in the IMG gene product name, with predicted proteins without essential active site residues assigned product names implying enzymatic activity (21 proteins, 2.8% of proteins inspected) or Enzyme Commission (E. C.) numbers (57 proteins, 7.7%). There were also 57 proteins (7.7%) assigned overly generic names and 78 proteins (10.6%) without E.C. numbers as part of the assigned name when a specific enzyme name and E. C. number were well-justified.ConclusionsThe patchy distribution of purine biosynthetic genes in archaea is consistent with a pathway that has been shaped by horizontal gene transfer, duplication, and gene loss. Our results indicate that manual curation can improve upon automated annotation for a small number of automatically-annotated proteins and can reveal a need to identify further pathway components even in well-studied pathways.ReviewersThis article was reviewed by Dr. Céline Brochier-Armanet, Dr Kira S Makarova (nominated by Dr. Eugene Koonin), and Dr. Michael Galperin.