Formylglycinamide Ribonucleotide Research Articles

Glutamine Antagonist GA-607 Causes a Dramatic Accumulation of FGAR which can be used to Monitor Target Engagement.

BackgroundMetabolomic analyses from our group and others have shown that tumors treated with glutamine antagonists (GA) exhibit robust accumulation of formylglycinamide ribonucleotide (FGAR), an intermediate in the de novo purine synthesis pathway. The increase in FGAR is attributed to the inhibition of the enzyme FGAR amidotransferase (FGAR-AT) that catalyzes the ATP-dependent amidation of FGAR to formylglycinamidine ribonucleotide (FGAM). While perturbation of this pathway resulting from GA therapy has long been recognized, no study has reported systematic quantitation and analyses of FGAR in plasma and tumors.ObjectiveHerein, we aimed to evaluate the efficacy of our recently discovered tumor-targeted GA prodrug, GA-607 (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido)hexanamido)-6-diazo-5-oxohexanoate), and demonstrate its target engagement by quantification of FGAR in plasma and tumors.MethodsEfficacy and pharmacokinetics of GA-607 were evaluated in a murine EL4 lymphoma model followed by global tumor metabolomic analysis. Liquid chromatography-mass spectrometry (LC-MS) based methods employing the ion-pair chromatography approach were developed and utilized for quantitative FGAR analyses in plasma and tumors.ResultsGA-607 showed preferential tumor distribution and robust single-agent efficacy in a murine EL4 lymphoma model. While several metabolic pathways were perturbed by GA-607 treatment, FGAR showed the highest increase qualitatively. Using our newly developed sensitive and selective LC-MS method, we showed a robust >80- and >10-fold increase in tumor and plasma FGAR levels, respectively, with GA-607 treatment.ConclusionThese studies describe the importance of FGAR quantification following GA therapy in cancer and underscore its importance as a valuable pharmacodynamic marker in the preclinical and clinical development of GA therapies.

Abstract 3980: Dual targeting mitochondrial and cytosolic one-carbon metabolism via the proton-coupled folate transporter with novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolates

Abstract Cellular one-carbon (1C) metabolism generates a host of metabolites especially critical to cancer cells. Classical 1C inhibitors often primarily target a single enzyme, commonly leading to drug resistance and necessitating the development of novel compounds targeting multiple enzymes. We synthesized several novel 5-substituted pyrrolo[3,2-d]pyrimidine compounds (AGF291, AGF320, and AGF347) with transport by the proton-coupled folate transporter (PCFT), a folate transporter with a narrow physiological niche, but commonly expressed in many solid tumors. These analogs showed substantial inhibition in proliferation assays with Chinese hamster ovary sublines engineered to individually express PCFT or the ubiquitously-expressed reduced folate carrier (the major tissue folate transporter), as well as in the PCFT-expressing human cancer cell lines H460 (large cell lung carcinoma), HCT-116 (colorectal carcinoma), and MIA PaCa-2 (pancreatic ductal adenocarcinoma). Full abrogation of inhibitory effects for all compounds required co-treatment with both adenosine and glycine, suggesting dual-targeting of cytosolic de novo purine biosynthesis and mitochondrial 1C/glycine metabolism. For cytosolic de novo purine biosynthesis, lack of protection by 5-aminoimidazole-4-carboxamide (AICA) suggested AICA ribonucleotide formyltransferase (AICARFTase), the second folate-dependent enzyme, as the likely enzyme target. AICARFTase inhibition was confirmed in intact H460 and HCT-116 cells by measuring accumulation of AICAR by targeted metabolomics. Targeting of mitochondrial enzymes in H460 and HCT-116 cells was suggested by selective inhibition of incorporation of [3-14C]serine over [14C]formate into the purine intermediate [14C]formyl glycinamide ribonucleotide. [2,3,3-2H]Serine isotope label scrambling analysis confirmed the mitochondrial target to be serine hydroxymethyltransferase2 (SHMT2). SHMT2 is a potentially promising anticancer drug target as its expression is highly correlated with the malignant phenotype across a broad spectrum of cancers beyond lung and colon, including breast, glioma, and liver. Indeed, our initial in-vivo studies with AGF291 suggest potent efficacy toward both MIA PaCa-2 and H460 xenograft mouse models. Our studies demonstrate the potential for tumor-selective dual targeting of both mitochondrial and cytosolic 1C metabolism to overcome resistance to classical 1C inhibitors. Citation Format: Aamod S. Dekhne, Gregory S. Ducker, Josephine Frühauf, Khushbu Shah, Md. Junayed Nayeen, Adrianne Wallace-Povirk, Carrie O'Connor, Zhanjun Hou, Lisa Polin, Aleem Gangjee, Joshua D. Rabinowitz, Larry H. Matherly. Dual targeting mitochondrial and cytosolic one-carbon metabolism via the proton-coupled folate transporter with novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3980.

Abstract 3983: Tumor-targeting with novel dual-targeted 6-substituted thieno[2,3-d]pyrimidine antifolates via cellular uptake by folate receptor α, and inhibition of de novo purine nucleotide biosynthesis

Abstract Epithelial ovarian cancer (EOC) is the deadliest gynecologic cancer, with limited progress in targeted therapy or in combating drug resistance. Reflecting this, there is an urgent need to develop novel inhibitors with minimal toxicity. EOC is associated with increasing expression of folate receptor alpha (FRα) with increasing stage and grade of disease. Of particular interest are targeted therapeutics toward FRα which spare transport by the ubiquitously expressed reduced folate carrier (RFC). FRα is a viable target for tumor selectivity with limited expression in normal tissues, where it is only expressed on apical membranes. We previously described 6-substituted thieno[2,3-d]pyrimidine (6TP) benzoyl antifolates with bridge lengths of 2-8 carbons as potent and selective inhibitors of tumor cells including EOCs that express FRs. The most potent compound (AGF50) included a 4-carbon bridge and showed FRα selectivity and inhibition of de novo purine biosynthesis. We synthesized AGF50 analogs with various aryl side-chain modifications (e.g., thiophene, furan, pyridine) including 2'fluorine substitutions. Toward isogenic Chinese hamster ovary cell lines individually expressing FRα, RFC and the proton-coupled folate transporter (PCFT), the 6TP analogs were completely selective toward FRα over RFC and PCFT, manifested as inhibition of cell proliferation with nmolar IC50s. Activity of this series extended to KB human tumor cells and human EOC cell lines (e.g., IGROV1). Drug effects were abolished with excess folic acid, confirming FRα uptake. Adenosine completely protected cells from the inhibitory effects of 6TPs in vitro, suggesting inhibition of one-carbon transfers mediated by glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) and/or 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferase (AICARFTase), leading to synthesis of purine nucleotides. Protection from 6TPs by AICA was incomplete (in contrast to a “pure” GARFTase inhibitor AGF94), suggesting inhibition of AICARFTase. Whereas AGF94 potently inhibited GARFTase (>98%) in KB cells, reflected in [14C]glycine incorporation into [14C]formyl GAR, inhibition by the 6TPs was incomplete (75-90%) up to 1 μM drug. AICARFTase inhibition was confirmed by measuring accumulation of AICA ribonucleotide (ZMP), although this varied for different 6TP analogs. Our studies establish an important structure-activity relationship for novel FRα-targeted 6TPs with aromatic side-chain modifications and imply the therapeutic potential of targeting AICARFTase for EOC. Citation Format: Adrianne Wallace-Povirk, Nian Tong, Carrie O'Connor, Zhanjun Hou, Aleem Gangjee, Larry Matherly, Xilin Zhou. Tumor-targeting with novel dual-targeted 6-substituted thieno[2,3-d]pyrimidine antifolates via cellular uptake by folate receptor α, and inhibition of de novo purine nucleotide biosynthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3983.

An efficient and practical synthesis of formylglycinamide ribonucleotide (FGAR)

GRAPHICAL ABSTRACT

Communication between the two catalytic domains of formylglycinamide ribonucleotide amidotransferase

Communication between the two catalytic domains of formylglycinamide ribonucleotide amidotransferase

Crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus, Sulfolobus tokodaii and Methanocaldococcus jannaschii.

The crystal structures of a subunit of the formylglycinamide ribonucleotide amidotransferase, PurS, from Thermus thermophilus, Sulfolobus tokodaii and Methanocaldococcus jannaschii were determined and their structural characteristics were analyzed. For PurS from T. thermophilus, two structures were determined using two crystals that were grown in different conditions. The four structures in the dimeric form were almost identical to one another despite their relatively low sequence identities. This is also true for all PurS structures determined to date. A few residues were conserved among PurSs and these are located at the interaction site with PurL and PurQ, the other subunits of the formylglycinamide ribonucleotide amidotransferase. Molecular-dynamics simulations of the PurS dimer as well as a model of the complex of the PurS dimer, PurL and PurQ suggest that PurS plays some role in the catalysis of the enzyme by its bending motion.

Abstract 4791: Targeting ovarian cancer with novel 6-substituted pyrrolo [2,3-d] pyrimidine fluorinated antifolates via cellular uptake by folate receptor á and the proton-coupled folate transporter

Abstract The proton coupled folate transporter (PCFT) is a proton-folate symporter with an acidic pH optimum, while folate receptor (FR) á delivers folate substrates into the cell via endocytosis. FRá and PCFT are highly expressed in ovarian cancer. PCFT transport is favored by acidic pH, including pHs associated with the acidic tumor microenvironment. We previously synthesized a novel class of 6-substituted pyrrolo[2,3-d]pyrimidine thiophene antifolates with 4- and 3-carbon bridge lengths, designated AGF117 and AGF150, targeted to FRá and PCFT, but not the ubiquitously expressed reduced folate carrier (RFC). To increase antitumor specificity and activity, reflecting multi-polar interactions with cellular targets, we synthesized fluorinated analogs of AGF117 and AGF150, designated AGF278 and AGF283. In the presence of a physiologic concentration of reduced folate, AGF278 and AGF283 potently inhibited proliferation of Chinese hamster ovary (CHO) cells engineered to express PCFT, but not RFC or FRá (R2/PCFT4) (IC50s of 6 and 1.5 nM, respectively), or FRá without PCFT or RFC (RT16) (IC50s of 4.8 and 5.4 nM). AGF278 and AGF283 also inhibited growth of IGROV1 (IC50s of 1.5 and 11 nM, respectively) and SKOV3 (IC50s of 37.8 and 30.2 nM, respectively) ovarian cancer cells. We measured [3H]methotrexate uptake in R2/PCFT4 CHO cells in the presence of AGF278 and AGF283; AGF278 and AGF283 potently inhibited PCFT transport with increased binding over AGF117 and AGF150, respectively, as reflected in Ki values. Analogous to AGF117 and AGF150, glycinamide ribonucleotide (GAR) formyltransferase (GARFTase), the first folate-dependent enzyme in de novo purine biosynthesis, was implicated as the cellular target for AGF278 and AGF283 as adenosine (10 ìM) and 5-aminoimidazole-4-carboxamide (320 ìM) protected IGROV1 cells from growth inhibition. GARFTase inhibition by AGF278 and AGF283 was confirmed by an in situ GARFTase assay which measures incorporation of [14C]glycine into formyl GAR, the GARFTase product, with potencies similar to those of the parent molecules. Preliminary in vivo studies with AGF278 using SKOV3 tumor xenografts in SCID mice established antitumor efficacy. Our results suggest that novel fluorinated 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates may offer significant promise for treating ovarian cancer, reflecting their selective cellular uptake by PCFT and FRá and potent inhibition of de novo purine biosynthesis. Additional utility of our fluorine-substituted analogs lies in their potential for positron emission tomography imaging of PCFT-expressing tumors. Citation Format: Mike R. Wilson, Manasa P. Ravindraa, Adrianne Wallace-Povirk, Lisa Polin, Zhanjun Hou, Aleem Gangjee, Larry H. Matherly. Targeting ovarian cancer with novel 6-substituted pyrrolo [2,3-d] pyrimidine fluorinated antifolates via cellular uptake by folate receptor á and the proton-coupled folate transporter. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4791.

Determination of the formylglycinamide ribonucleotide amidotransferase ammonia pathway by combining 3D-RISM theory with experiment.

Molecular tunnels in enzyme systems possess variable architecture and are therefore difficult to predict. In this work, we design and apply an algorithm to resolve the pathway followed by ammonia using the bifunctional enzyme formylglycinamide ribonucleotide amidotransferase (FGAR-AT) as a model system. Though its crystal structure has been determined, an ammonia pathway connecting the glutaminase domain to the 30 Å distal FGAR/ATP binding site remains elusive. Crystallography suggested two purported paths: an N-terminal-adjacent path (path 1) and an auxiliary ADP-adjacent path (path 2). The algorithm presented here, RismPath, which enables fast and accurate determination of solvent distribution inside a protein channel, predicted path 2 as the preferred mode of ammonia transfer. Supporting experimental studies validate the identity of the path, and results lead to the conclusion that the residues in the middle of the channel do not partake in catalytic coupling and serve only as channel walls facilitating ammonia transfer.

Importance of Hydrophobic Cavities in Allosteric Regulation of Formylglycinamide Synthetase: Insight from Xenon Trapping and Statistical Coupling Analysis

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) is a 140 kDa bi-functional enzyme involved in a coupled reaction, where the glutaminase active site produces ammonia that is subsequently utilized to convert FGAR to its corresponding amidine in an ATP assisted fashion. The structure of FGAR-AT has been previously determined in an inactive state and the mechanism of activation remains largely unknown. In the current study, hydrophobic cavities were used as markers to identify regions involved in domain movements that facilitate catalytic coupling and subsequent activation of the enzyme. Three internal hydrophobic cavities were located by xenon trapping experiments on FGAR-AT crystals and further, these cavities were perturbed via site-directed mutagenesis. Biophysical characterization of the mutants demonstrated that two of these three voids are crucial for stability and function of the protein, although being ∼20 Å from the active centers. Interestingly, correlation analysis corroborated the experimental findings, and revealed that amino acids lining the functionally important cavities form correlated sets (co-evolving residues) that connect these regions to the amidotransferase active center. It was further proposed that the first cavity is transient and allows for breathing motion to occur and thereby serves as an allosteric hotspot. In contrast, the third cavity which lacks correlated residues was found to be highly plastic and accommodated steric congestion by local adjustment of the structure without affecting either stability or activity.

Abstract 5494: Novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate regioisomers target folate receptor alpha positive ovarian cancer cells via inhibition of de novo purine nucleotide biosynthesis.

Abstract Epithelial ovarian cancers (EOC) represent a unique treatment challenge as over 50% of patients are diagnosed with advanced stage disease when surgical and chemotherapeutic interventions prove futile. Approximately 90% of EOC are folate receptor (FR) α positive and FR levels correlate with stage and grade. FRα-targeted therapies represent a rational treatment option for patients with advanced EOC whose treatments fail. There are three primary mechanisms of folate transport in mammalian cells, the reduced folate carrier (RFC), the proton-coupled folate transporter (PCFT) and FRs. RFC is the major transporter for folates and is expressed ubiquitously in normal tissues and tumors. Hence, developing novel antifolates that bypass cellular accumulation by RFC should reduce dose-limiting toxicities. PCFT is expressed in many solid tumors including ovarian cancer and is active at acidic pH. Previous studies established that a novel 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolate with an isosteric thienoyl substitution and a three carbon bridge (AG94) showed selective cellular uptake by FRα and PCFT over RFC. [1,3]Thienoyl regioisomers (AG150 and AG154) of AG94 showed subnanomolar potencies toward both KB nasopharyngeal carcinoma (IC50s = 0.1 and 0.09 nM, respectively) and IGROV1 ovarian carcinoma (IC50s of 0.64 and 0.1nM, respectively). While AG150 and AG154 were substrates for both PCFT and FRs, like AG94, their inhibitory effects at low drug concentrations were mediated primarily via FRα, as indicated by their capacities to bind with high affinities to FRα and protection by excess folic acid. Nucleoside protection assays with adenosine and 5-amino-4-imidazole carboxamide established that the first folate-dependent enzyme in the de novo purine biosynthesis pathway, glycinamide ribonucleotide (GAR) formyltransferase (GARFT), was the primary intracellular target for AG150 and AG154. GARFT targeting was confirmed by in situ metabolic labeling with [14C]glycine and measurement of accumulated [14C]formyl GAR in KB cells, for which IC50s were 1.35 and 1.46 nM, respectively. The purine salvage enzyme methylthioadenosine phosphorylase (MTAP) is frequently co-deleted with CDKN2a in solid tumors including ovarian cancer. Growth inhibitory effects of AG150 and AG154 were reversed in the presence of methylthioadenosine in MTAP-deficient IGROV1 cells transfected with MTAP cDNA. Our results demonstrate potent anti-tumor activities of the 6-pyrrolo[2,3-d]pyrimidine thienoyl regioisomers of AG94, AG150 and AG154, associated with their FRα-mediated cellular uptake and inhibition of GARFT in de novo purine biosynthesis. Preservation of purine salvage including MTAP is likely an important cellular determinant of antitumor efficacy for this class of novel tumor-targeted agents. Citation Format: Shermaine K. Mitchell-Ryan, Lei Wang, Steven Orr, Sita Kugel, Christina Cherian, Aleem Gangjee, Larry H. Matherly. Novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate regioisomers target folate receptor alpha positive ovarian cancer cells via inhibition of de novo purine nucleotide biosynthesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5494. doi:10.1158/1538-7445.AM2013-5494

Murine gammaherpesvirus 68 ORF75c contains ubiquitin E3 ligase activity and requires PML SUMOylation but not other known cellular PML regulators, CK2 and E6AP, to mediate PML degradation

All gammaherpsviruses encode at least one gene related to the cellular formylglycinamide ribonucleotide amidotransferase (FGARAT) enzyme but their biological roles are relatively unknown. The murine gammaherpesvirus 68 (MHV68) vFGARAT, ORF75c, mediates a proteasome-dependent degradation of the antiviral promyelocytic leukemia (PML) protein by an unknown mechanism, which is addressed in this study. We found that ORF75c interacts weakly with PML and SUMO-modified forms of PML are important for its degradation by ORF75c. ORF75c-mediated PML degradation was not dependent on two known cellular regulators of PML stability, Casein kinase II (CK2) and human papilloma virus E6-associated protein (E6AP). Finally, ORF75c had self-ubiquitination activity in vitro and its expression increased levels of ubiquitinated PML in transfected cells. Taken together, the evidence accumulated in this study provides new insights into the function of a vFGARAT and is consistent with a model in which ORF75c could mediate direct ubiquitination of PML resulting in its degradation by the proteasome.

Formylglycinamide ribonucleotide amidotransferase fromSalmonella typhimurium: role of ATP complexation and the glutaminase domain in catalytic coupling

Formylglycinamide ribonucleotide (FGAR) amidotransferase (FGAR-AT) takes part in purine biosynthesis and is a multidomain enzyme with multiple spatially separated active sites. FGAR-AT contains a glutaminase domain that is responsible for the generation of ammonia from glutamine. Ammonia is then transferred via a channel to a second active site located in the synthetase domain and utilized to convert FGAR to formylglycinamidine ribonucleotide (FGAM) in an adenosine triphosphate (ATP) dependent reaction. In some ammonia-channelling enzymes ligand binding triggers interdomain signalling between the two diverse active centres and also assists in formation of the ammonia channel. Previously, the structure of FGAR-AT from Salmonella typhimurium containing a glutamyl thioester intermediate covalently bound in the glutaminase active site was determined. In this work, the roles played by various ligands of FGAR-AT in inducing catalytic coupling are investigated. Structures of FGAR-AT from S. typhimurium were determined in two different states: the unliganded form and the binary complex with an ATP analogue in the presence of the glutamyl thioester intermediate. The structures were compared in order to decipher the roles of these two states in interdomain communication. Using a process of elimination, the results indicated that binding of FGAR is most likely to be the major mechanism by which catalytic coupling occurs. This is because conformational changes do not occur either upon formation of the glutamyl thioester intermediate or upon subsequent ATP complexation. A model of the FGAR-bound form of the enzyme suggested that the loop in the synthetase domain may be responsible for initiating catalytic coupling via its interaction with the N-terminal domain.

Abstract 2528: Therapeutic targeting of a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate to human solid tumors based on selective uptake by PCFT

Abstract The proton-coupled folate transporter (PCFT) is a proton-folate symporter with an acidic pH optimum, such that PCFT may provide a mechanism for targeting cytotoxic antifolates to tumors, based on their acidic microenvironments (pH 6.2-6.8). PCFT transcripts and protein are high in human solid tumor tissues and cell lines (especially hepatic and ovarian tumors) and limited in most human normal tissues. We identified a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate (AG71) that is a selective transport substrate for PCFT over the ubiquitously expressed reduced folate carrier (RFC) and a potent inhibitor of the de novo purine nucleotide biosynthetic enzyme glycinamide ribonucleotide (GAR) formyl transferase (GARFTase). Both AG71 and the clinically relevant antifolate pemetrexed (Pmx) were potently inhibitory toward proliferation of a HeLa subline (R1-11-PCFT4) that lacked RFC expression and was engineered to ectopically express physiologically relevant PCFT levels. Unlike Pmx, AG71 did not inhibit proliferation of R1-11-RFC6 cells which express physiologically relevant RFC levels but no PCFT. In colony-forming assays at pH 6.8, AG71 inhibited colony formation of R1-11-PCFT4 cells in a dose- and time-dependent manner, comparable to Pmx. We measured uptake of [3H]AG71 in R1-11-PCFT4 cells and found that transport was optimal at pH 5.5 but still significant at pH 6.8. At pH 6.8, AG71 was metabolized by folylpoly-γ-glutamate synthase to AG71 polyglutamates, as detected by HPLC. GARFTase was validated as a major intracellular target with an in situ GARFTase activity assay at pH 6.8 involving [14C]glycine incorporation into [14C]formyl GAR. In HepG2 human hepatocellular carcinoma cells, PCFT levels were elevated and [3H]AG71 was transported at pH 6.8, whereupon it was extensively metabolized to polyglutamates. By proliferation assays, AG71 was potently inhibitory (IC50 of 227.5 nM) toward HepG2 cells. To test in vivo efficacy of AG71, SCID mice were implanted with subcutaneous HepG2 tumors and treated with AG71 (180 mg/kg × 3d). Compound AG71 was highly active against HepG2 tumors. In conclusion, we have established the expression patterns and pH-specific transport for PCFT in human solid tumor cells and demonstrated that the novel PCFT-targeted antifolate AG71 can be internalized into tumor cells by PCFT leading to significant growth delay of HepG2 xenografts. Our findings offer exciting new therapeutic possibilities for selectively targeting chemotherapy to tumors with novel antifolates based on their acidic microenvironments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2528. doi:10.1158/1538-7445.AM2011-2528

Formylglycinamide Ribonucleotide Amidotransferase from Thermotoga maritima: Structural Insights into Complex Formation

In the fourth step of the purine biosynthetic pathway, formyl glycinamide ribonucleotide (FGAR) amidotransferase, also known as PurL, catalyzes the conversion of FGAR, ATP, and glutamine to formyl glycinamidine ribonucleotide (FGAM), ADP, Pi, and glutamate. Two forms of PurL have been characterized, large and small. Large PurL, present in most Gram-negative bacteria and eukaryotes, consists of a single polypeptide chain and contains three major domains: the N-terminal domain, the FGAM synthetase domain, and the glutaminase domain, with a putative ammonia channel located between the active sites of the latter two. Small PurL, present in Gram-positive bacteria and archaea, is structurally homologous to the FGAM synthetase domain of large PurL, and forms a complex with two additional gene products, PurQ and PurS. The structure of the PurS dimer is homologous with the N-terminal domain of large PurL, while PurQ, whose structure has not been reported, contains the glutaminase activity. In Bacillus subtilis, the formation of the PurLQS complex is dependent on glutamine and ADP and has been demonstrated by size-exclusion chromatography. In this work, a structure of the PurLQS complex from Thermotoga maritima is described revealing a 2:1:1 stoichiometry of PurS:Q:L, respectively. The conformational changes observed in TmPurL upon complex formation elucidate the mechanism of metabolite-mediated recruitment of PurQ and PurS. The flexibility of the PurS dimer is proposed to play a role in the activation of the complex and the formation of the ammonia channel. A potential path for the ammonia channel is identified.

Complexed Structures of Formylglycinamide Ribonucleotide Amidotransferase from Thermotoga maritima Describe a Novel ATP Binding Protein Superfamily,

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP-dependent synthesis of formylglycinamidine ribonucleotide (FGAM) from formylglycinamide ribonucleotide (FGAR) and glutamine in the fourth step of the purine biosynthetic pathway. FGAR-AT is encoded by the purL gene. Two types of PurL have been detected. The first type, found in eukaryotes and Gram-negative bacteria, consists of a single 140 kDa polypeptide chain and is designated large PurL (lgPurL). The second type, small PurL (smPurL), is found in archaea and Gram-positive bacteria and consists of an 80 kDa polypeptide chain. SmPurL requires two additional gene products, PurQ and PurS, for activity. PurL is a member of a protein superfamily that contains a novel ATP-binding domain. Structures of several members of this superfamily are available in the unliganded form. We determined five different structures of FGAR-AT from Thermotoga maritima in the presence of substrates, a substrate analogue, and a product. These complexes have allowed a detailed description of the novel ATP-binding motif. The availability of a ternary complex enabled mapping of the active site, thus identifying potential residues involved in catalysis. The complexes show a conformational change in the active site compared to the unliganded structure. Surprising discoveries, an ATP molecule in an auxiliary site of the protein and the conformational changes associated with its binding, provoke speculation about the regulatory role of the auxiliary site in formation of the PurLSQ complex as well as the evolutionary relationship of PurLs from different organisms.

Crystal structure of phosphoribosylformyl‐glycinamidine synthase II, PurS subunit (TM1244) from Thermotoga maritima at 1.90 Å resolution

Crystal structure of phosphoribosylformyl‐glycinamidine synthase II, PurS subunit (TM1244) from <i>Thermotoga maritima</i> at 1.90 Å resolution

Crystal structure of phosphoribosylformylglycinamidine synthase II (smPurL) from Thermotoga maritima at 2.15 Å resolution

Crystal structure of phosphoribosylformylglycinamidine synthase II (smPurL) from <i>Thermotoga maritima</i> at 2.15 Å resolution

A Model for the Bacillus subtilis Formylglycinamide Ribonucleotide Amidotransferase Multiprotein Complex,

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), ATP, and glutamine to formylglycinamidine ribonucleotide (FGAM), ADP, P(i), and glutamate in the fourth step of the purine biosynthetic pathway. PurL exists in two forms: large PurL (lgPurL) is a single chain, multidomain enzyme of about 1300 amino acids, whereas small PurL (smPurL) contains about 800 amino acids but requires two additional gene products, PurS and PurQ, for activity. smPurL contains the ATP and FGAR binding sites, PurQ is a glutaminase, and the function of PurS is just now becoming understood. We determined the structure of Bacillus subtilis PurS in two different crystal forms P2(1) and C2 at 2.5 and 2.0 A resolution, respectively. PurS forms a tight dimer with a central six-stranded beta-sheet flanked by four helices. In both the P2(1) and the C2 crystal forms, the quaternary structure of PurS is a tetramer. The concave faces of the PurS dimers interact via the C-terminal region to form a twelve-stranded beta-barrel with a hydrophilic core. We used the structure of PurS together with the structure of lgPurL from Salmonella typhimurium to construct a model of the PurS/smPurL/PurQ complex. The HisH (glutaminase) domain of imidazole glycerol phosphate synthetase was used as an additional model of PurQ. The model shows stoichiometry of 2PurS/smPurL/PurQ using a PurS dimer or 4PurS/2smPurL/2PurQ using a PurS tetramer. Both models place key conserved residues at the ATP/FGAR binding site and at a structural ADP binding site. The homology model is consistent with biochemical studies on the reconstituted complex.

The formylglycinamide ribonucleotide amidotransferase complex from Bacillus subtilis: metabolite-mediated complex formation.

Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP- and glutamine-dependent formation of formylglycinamidine ribonucleotide, ADP, P(i), and glutamate in the fourth step of de novo purine biosynthesis. Like all amidotransferases (ATs), FGAR-AT is proposed to channel ammonia between a glutaminase and AT domain. In Gram-negative bacteria and eukaryotes, FGAR-AT is a single approximately 140 kDa protein. In archae and Gram-positive bacteria, the FGAR-AT is formed from three proteins: PurS (10 kDa), PurQ (25 kDa, a glutaminase), and smPurL (80 kDa, an AT). This is the only known AT to require a third structural component (PurS) for activity. Here we report the first purification and biochemical characterization of a three-component AT from Bacillus subtilis. Efforts to isolate an intact FGAR-AT focused initially on coexpression of PurS, smPurL, and PurQ. However, all attempts to purify the complex resulted in separation of the constituent proteins. PurS, smPurL, and PurQ were therefore separately expressed and purified to homogeneity. PurQ had a glutaminase activity of 0.002 s(-1), and smPurL had an ammonia-dependent AT activity of 0.044 s(-1). Reconstitution of PurS, smPurL, and PurQ at a ratio of 2:1:1 gave an activity of 2.49 s(-1), similar to that previously reported for the Escherichia coli 140 kDa FGAR-AT (5.00 s(-1)). PurS was essential for the glutamine-dependent FGAR-AT activity. Surprisingly, activity was found to be absolutely dependent on the presence of Mg2+ and ADP, and a stable FGAR-AT complex of 2PurS/1smPurL/1PurQ was detected only in the presence of Mg2+, ADP, and glutamine. The implications of these observations are discussed with respect to ammonia channeling.

Linkage between toxin production and purine biosynthesis in Clostridium difficile.

The production of toxins A and B by Clostridium difficile was greatly enhanced under biotin-limited conditions, in which a 140-kDa protein was expressed strongly. Gene cloning revealed that this protein was a homologue of formylglycinamidine ribonucleotide synthetase (FGAM synthetase, EC 6.3.5.3), which is known as PurL in Escherichia coli and catalyses the fourth step of the de novo purine biosynthesis pathway. This enzyme consisted of a single polypeptide, although FGAM synthetases of gram-positive bacteria usually consist of two subunits. Inhibition of the enzymic activity of C. difficile PurL by O-diazoacetyl-L-serine (azaserine) resulted in enhanced toxin B production even in biotin-sufficient conditions. In contrast, blockade of the preceding step of the PurL catalysing step by sulfamethoxazole inhibited toxin B production almost completely. These results suggest that accumulation of formylglycinamide ribonucleotide (FGAR), a substrate of FGAM synthetase, enhances toxin production by C difficile and depletion of FGAR reduces toxin production.