Aminoimidazole Carboxamide Ribonucleotide Formyltransferase Research Articles

Analysis of substrate binding in individual active sites of bifunctional human ATIC

Aminoimidazolecarboxamide ribonucleotide formyl transferase (AICARFT): Inosine monophosphate cyclohydrolase (IMPCH, collectively called ATIC) is a bifunctional enzyme that catalyses the penultimate and final steps in the purine de novo biosynthesis pathway. The bifunctional protein is dimeric and each monomer contains two different active sites both of which are capable of binding nucleotide substrates, this means to a potential total of four distinct binding events might be observed. Within this work we used a combination of site-directed and truncation mutants of ATIC to independently investigate the binding at these two sites using calorimetry. A single S10W mutation is sufficient to block the IMPCH active site allowing investigation of the effects of mutation on ligand binding in the AICARFT active site. The majority of nucleotide ligands bind selectively at one of the two active sites with the exception of xanthosine monophosphate, XMP, which, in addition to binding in both AICARFT and IMPCH active sites, shows evidence for cooperative binding with communication between symmetrically-related active sites in the two IMPCH domains. The AICARFT site is capable of independently binding both nucleotide and folate substrates with high affinity however no evidence for positive cooperativity in binding could be detected using the model ligands employed in this study.

Reduced Folate Carrier and Folylpolyglutamate Synthetase, but not Thymidylate Synthase Predict Survival in Pemetrexed-Treated Patients Suffering from Malignant Pleural Mesothelioma

Malignant mesothelioma is a highly aggressive tumor arising from mesothelial-lined surfaces, most often in the pleura cavities. Antifolates belong to the most effective cytotoxic drugs for malignant pleural mesothelioma (MPM) treatment. Pemetrexed is an antifolate inhibiting different folate pathway genes (thymidylate synthase [TS], dihydrofolate reductase, glycinamide ribonucleotide formyltransferase [GARFT], and aminoimidazole carboxamide ribonucleotide formyltransferase, [AICARFT]). Increased activity of pemetrexed occurs by folylpolyglutamate synthetase (FPGS), intracellular transport by reduced folate carrier (RFC). The aim of the study was to explore potential correlations between TS, GARFT, AICARFT, RFC, and FPGS levels in MPM and associations with clinical benefit from pemetrexed treatment. Samples from 63 patients were tested using immunohistochemistry (IHC) and quantitative polymerase chain reaction(qPCR) for expression levels of TS, GARFT, AICARFT, RFC, and FPGS. Clinical data were evaluated to determine associations between efficacy of pemetrexed and enzyme expression levels. Evaluation of expression levels was done through TaqMan-based qPCR, and IHC was evaluated semiquantitatively by using the H-score. qPCR analysis showed no difference in expression pattern of GARFT and AICARFT. IHC analysis revealed a heterogeneous staining pattern for all the enzymes. No significant association was found between TS expression and survival or objective response of the tumors after pemetrexed treatment. FPGS (p = 0.0111) and RFC (p = 0.0088) mRNA expression levels were strongly associated with overall survival in these patients. Our results reveal that in pemetrexed-treated MPMs TS expression levels have no influence on patient outcome. Furthermore, GARFT and AICARFT were homogeneously expressed in the patient samples. Folate uptake mechanisms by RFC and activation by FPGS were associated with clinical benefit from pemetrexed treatment.

Expression of folate pathway regulators and pemetrexed (pem) activity in patients (pts) with advanced non-small cell lung cancer (NSCLC).

7585 Background: Thymidylate synthase (TS) expression has been reported to predict pem activity in pts with advanced NSCLC. Besides TS, pem inhibits multiple enzymes of the folate pathway including dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyltransferase (GART) and aminoimidazole carboxamide ribonucleotide formyltransferase (AICAR). Aim of present study was to investigate whether these or other biomarkers influence pem activity in NSCLC pts. Methods: Advanced pretreated NSCLC pts who received at least two cycles of single agent pem were considered eligible if they had available tumor tissue to assess at least one of the proposed biomarkers. TS, DHFR, GART and AICAR protein expression was assessed by immunohistochemistry (IHC) in FFPE tumor samples. Pts were grouped as IHC+ and IHC– according to staining intensity. Additionally, presence of EGFR and KRAS mutations was investigated. Results: Ninety-six pts were included in the study. The majority of subjects was male (72%), smokers (93%), with adenocarcinoma (72%). Response rate (RR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS) were 12%, 47%, 2.3 and 9.5 months, respectively. Most pts resulted TS-(72%), AICAR-(82%), DHFR+ (68%), GART- (61%). EGFR and KRAS mutations were identified in 15% and 25% of assessable cases. None of the biomarkers was significantly associated with pts characteristics (gender, histology, smoking status), nor with RR or DCR. GART- pts experienced longer PFS when compared with the GART+ group (HR 0.56, p=.052), while no difference was observed according to TS, DHFR and AICAR status. A significantly longer OS was observed for TS- (HR 0.52, p=.012), GART- (HR 0.50, p=.037) and AICAR- (HR 0.40, p=.028) pts. No significant difference in the distribution of EGFR mutant pts receiving EGFR TKIs after pem failure was observed between IHC+ and IHC- groups. Conclusions: Low TS, GART and AICAR protein expression predicts significantly prolonged survival in single agent pem-treated pts with advanced NSCLC. The lack of PFS difference according to TS and AICAR status suggests prognostic rather than predictive relevance for these biomarkers.

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.

Pemetrexed Indirectly Activates the Metabolic Kinase AMPK in Human Carcinomas

The chemotherapeutic drug pemetrexed, an inhibitor of thymidylate synthase, has an important secondary target in human leukemic cells, aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate-dependent enzyme of purine biosynthesis. The purine intermediate aminoimidazolecarboxamide ribonucleotide (ZMP), which accumulates behind this block, transmits an inhibitory signal to the mTORC1 complex via activation of the cellular energy sensor AMP-activated kinase (AMPK). Given that the PI3K-AKT-mTOR pathway is frequently deregulated during carcinogenesis, we asked whether the indirect activation of AMPK by pemetrexed offers an effective therapeutic strategy for carcinomas with defects in this pathway. Activation of AMPK by ZMP in pemetrexed-treated colon and lung carcinoma cells and the downstream consequences of this activation were strikingly more robust than previously seen in leukemic cells. Genetic experiments demonstrated the intermediacy of AICART inhibition and the centrality of AMPK activation in these effects. Whereas AMPK activation resulted in marked inhibition of mTORC1, other targets of AMPK were phosphorylated that were not mTORC1-dependent. Whereas AMPK activation is thought to require AMPKα T172 phosphorylation, pemetrexed also activated AMPK in carcinoma cells null for LKB1, the predominant AMPKα T172 kinase whose deficiency is common in lung adenocarcinomas. Like rapamycin analogs, pemetrexed relieved feedback suppression of PI3K and AKT, but the prolonged accumulation of unphosphorylated 4E-BP1, a tight-binding inhibitor of cap-dependent translation, was seen following AMPK activation. Our findings indicate that AMPK activation by pemetrexed inhibits mTORC1-dependent and -independent processes that control translation and lipid metabolism, identifying pemetrexed as a targeted therapeutic agent for this pathway that differs significantly from rapamycin analogs.

Abstract 5086: Pemetrexed induces a strong activation of AMPK in human carcinoma cells, even in the absence of LKB1

Abstract AMP-activated protein kinase (AMPK) is a major cellular energy sensor, functioning as a regulator of signaling cascades controlling protein, lipid, and carbohydrate metabolism. Because of the dependence of many tumors on the deregulation of the PI3K-AKT-mTOR signaling cascade, the discovery of AMPK activators is an attractive approach for cancer therapeutics, aimed at reinstating control over cellular metabolic functions altered during carcinogenesis rather than inhibiting cellular targets. The antifolate pemetrexed is FDA approved for first line treatment of mesothelioma and non-small cell lung cancer (NSCLC). While pemetrexed acts primarily as a thymidylate synthase inhibitor, we discovered that the drug is also an inhibitor of aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate-dependent enzyme of de novo purine synthesis. The inhibition of AICART by pemetrexed resulted in a marked accumulation of the reaction substrate, ZMP, a known allosteric activator of AMPK in several human carcinoma cell lines. The mammalian target of rapamycin complex 1 (mTORC1) was inhibited following pemetrexed treatment, as determined by hypophosphorylation of S6K1 and 4EBP1, and this effect was dependent on AMPK activation. Co-administration of GSK690693, a pan AKT inhibitor prolonged mTORC1 inhibition following pemetrexed treatment and offset the consequences of negative feedback activation of mTORC1 through PI3-kinase and mTORC2 activity. Interestingly, cells insensitive to mTORC1 inhibition following AMPK activation retained sensitivity to the secondary effect of pemetrexed, apparently due to inhibition of lipid synthesis through phosphorylation of acetyl-CoA carboxylase by AMPK. We concluded that the growth inhibitory effect of pemetrexed following AMPK activation in human carcinoma cells was due to a combined effect of inhibition of mTORC1 signaling and lipogenesis, both of which were mediated by activation of AMPK. Somewhat surprisingly, several cell lines null for LKB1, the major upstream kinase of AMPK, retained sensitivity to the secondary effects of pemetrexed mediated through AMPK activation. This suggests that the effect of pemetrexed on AMPK activity will be preserved in tumors lacking LKB1, a common genotype of human NSCLC. Hence, pemetrexed exerts its effects on carcinoma cells by both inhibition of traditional antifolate targets and by a novel mechanism, activation of AMPK. (Supported in part by NIH Grant CA-27605) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5086.

Therapeutics by Cytotoxic Metabolite Accumulation: Pemetrexed Causes ZMP Accumulation, AMPK Activation, and Mammalian Target of Rapamycin Inhibition

Pemetrexed represents the first antifolate cancer drug to be approved by the Food and Drug Administration in 20 years; it is currently in widespread use for first line therapy of mesothelioma and non-small cell lung cancer. Pemetrexed has more than one site of action; the primary site is thymidylate synthase. We now report that the secondary target is the downstream folate-dependent enzyme in de novo purine synthesis, aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART). The substrate of the AICART reaction, ZMP, accumulated in intact pemetrexed-inhibited tumor cells, identifying AICART as the step in purine synthesis that becomes rate-limiting after drug treatment. The accumulating ZMP causes an activation of AMP-activated protein kinase with subsequent inhibition of the mammalian target of rapamycin (mTOR) and hypophosphorylation of the downstream targets of mTOR that control initiation of protein synthesis and cell growth. We suggest that the activity of pemetrexed against human cancers is a reflection of its direct inhibition of folate-dependent target proteins combined with prolonged inhibition of the mTOR pathway secondary to accumulation of ZMP.

Molecular mechanisms underlying the enhanced sensitivity of thiopurine-resistant T-lymphoblastic cell lines to methyl mercaptopurineriboside

Methylmercaptopurine riboside (meMPR), a cellular metabolite of 6-mercaptopurine (6-MP), is a potent inhibitor of de novo purine synthesis (DNPS). Human MOLT4 T-lymphoblastic leukaemia cells that have acquired resistance to 6-MP or 6-thioguanine (6-TG) as a consequence of defective transport exhibit enhanced sensitivity to meMPR. HPLC-based analysis of the transport of meMPR revealed normal uptake of this compound by our thiopurine-resistant cell sublines, suggesting a route of transport distinct from that for 6-MP and 6-TG. Studies on the wild-type parental leukemic cells showed that adenosine, dipyridamole and nitrobenzylthioinosine inhibit uptake of meMPR to a significant extent, whereas Na + ions have no influence on this process. Transfection of these leukemic cells with small interference RNA molecules targeting the gene encoding the first member of the family of equiliberative nucleoside transporters (ENT1) strongly reduced the initial rate of meMPR transport. Our resistant cell lines exhibited 30–52% reductions ( p < 0.005) in their levels of mRNA encoding several proteins involved in de novo purine synthesis, i.e., aminoimidazole carboxamide ribonucleotide formyltransferase, glycinamide ribonucleotide transformylase and guanine monophosphate synthetase. Consequently, the rate of de novo purine synthesis in these resistant sublines was decreased by 50%. Furthermore, the levels of ribonucleoside triphosphates in these cells were significantly lower than in the non-resistant parental cells. In combination, a reduced rate of de novo purine synthesis together with low levels of ribonucleoside triphosphates can explain the enhanced sensitivity of our thiopurine-resistant cell lines to meMPR. In this manner, meMPR bypasses the mechanisms of resistance to thiopurines and is even more cytotoxic towards resistant than towards wild-type cells.

Pemetrexed in pancreatic cancer

New drugs are clearly needed for the treatment of advanced pancreatic cancer, a disease refractory to most chemotherapy. Pemetrexed, a novel antifolate, inhibits thymidylate synthase, dihydrofolate reductase, glycinamide ribonucleotide formyltransferase, and aminoimidazole carboxamide ribonucleotide formyltransferase. Pemetrexed is active against pancreatic cancer cell lines in vitro. Two partial responses in pancreatic cancer patients were observed in a phase I trial of pemetrexed. This led to a phase II trial of pemetrexed in patients with advanced pancreatic cancer. The objective response rate was 6%, 1-year survival rate was 28%, and the toxicities of therapy were mild. Pemetrexed is synergistic with gemcitabine in vitro. In a phase I trial, the pemetrexed/gemcitabine combination was broadly active and well tolerated. A phase II trial of this combination in 42 patients with advanced pancreatic cancer showed promising activity. A 520-patient international randomized phase III trial that compares the pemetrexed/gemcitabine combination with single-agent gemcitabine is currently accruing patients. Semin Oncol 29 (suppl 18):49-53. Copyright 2002, Elsevier Science (USA). All rights reserved.

Pemetrexed in pancreatic cancer.

New drugs are clearly needed for the treatment of advanced pancreatic cancer, a disease refractory to most chemotherapy. Pemetrexed, a novel antifolate, inhibits thymidylate synthase, dihydrofolate reductase, glycinamide ribonucleotide formyltransferase, and aminoimidazole carboxamide ribonucleotide formyltransferase. Pemetrexed is active against pancreatic cancer cell lines in vitro. Two partial responses in pancreatic cancer patients were observed in a phase I trial of pemetrexed. This led to a phase II trial of pemetrexed in patients with advanced pancreatic cancer. The objective response rate was 6%, 1-year survival rate was 28%, and the toxicities of therapy were mild. Pemetrexed is synergistic with gemcitabine in vitro. In a phase I trial, the pemetrexed/gemcitabine combination was broadly active and well tolerated. A phase II trial of this combination in 42 patients with advanced pancreatic cancer showed promising activity. A 520-patient international randomized phase III trial that compares the pemetrexed/gemcitabine combination with single-agent gemcitabine is currently accruing patients.

Pemetrexed disodium: a novel antifolate clinically active against multiple solid tumors.

Pemetrexed disodium (ALIMTA), "pemetrexed") is a novel, multi-targeted antifolate that has demonstrated promising clinical activity in a wide variety of solid tumors, including non-small cell lung, breast, mesothelioma, colorectal, pancreatic, gastric, bladder, cervix, and head and neck. Pemetrexed inhibits multiple folate-dependent enzymes involved in both purine and pyrimidine synthesis including thymidylate synthase, dihydrofolate reductase, glycinamide ribonucleotide formyltransferase, and aminoimidazole carboxamide ribonucleotide formyltransferase. As a single agent, pemetrexed exhibits a moderate toxicity profile at a dose of 500 mg/m(2) by 10-minute infusion once every 21 days with myelosuppression being the dose-limiting toxicity. Folic acid added to the diet in preclinical studies reduced toxicities while maintaining antitumor activity. Based on this observation and clinical toxicities, folic acid and vitamin B(12) dietary supplementation has been recently introduced into all ongoing trials. Studies combining pemetrexed with other active chemotherapeutic agents demonstrate that these combination therapies may become important treatment regimens in a variety of cancer types. Currently, pemetrexed phase III trials are ongoing in mesothelioma and non-small cell lung cancer with future trials planned to explore this unique multitargeted antifolate.

5,10-Methylenetetrahydro-5-deazafolic acid and analogs: synthesis and biological activities

The synthesis of 5,10-methylene-5-deazatetrahydrofolic acid (2), a stable, rigid analogue of 5,10-methylenetetrahydrofolate (1), is reported as a potential inhibitor of thymidylate synthase. The target compound was obtained by a Fisher-indole type cyclization of the hydrazone 16 from 2-amino-6-hydrazino-4-oxopyrimidine (10) and diethyl N-[4-(3-formyl-1-pyrrolyl)benzoyl]-L-glutamate (15) followed by catalytic reduction of the product 17. Similarly, modification of the Fisher-indole type cyclization of the appropriate hydrazone precursors 11 and 12 afforded the nonclassical analogues 3-amino-7,8,9-trimethyl-2H-pyrrolo[3',4':4,5]pyrido[2,3-d]pyrimidin-1- one (4) and 3-amino-8-benzyl-7,9-dimethyl-2H-pyrrolo[3',4':4,5]pyrido [2,3-d]pyrimidin-1-one (5), respectively. The target compound 2, its aromatic precursor 18, and the nonclassical analogue 4 were evaluated as inhibitors of the growth of Manca human lymphoma cells and also as inhibitors of human dihydrofolate reductase, human thymidylate synthase, glycinamide ribonucleotide formyltransferase, and aminoimidazole carboxamide ribonucleotide formyltransferase. Compound 18 showed weak inhibition of lymphoma cell growth (IC50 = 42 microM) and of AICAR formylTF (IC50 = 17 microM). Compounds 2 and 4 did not inhibit lymphoma cell growth or thymidylate synthase. The inactivity of 2 was attributed to its lack of flexibility leading to its inability to bind to thymidylate synthase.

Tetrahydrohomofolate polyglutamates as inhibitors of thymidylate synthase and glycinamide ribonucleotide formyltransferase in Lactobacillus casei

In order to determine the mechanism for the effects of homofolates on growth of Lactobacillus casei, polyglutamated derivatives of homofolate (HPteGlu), dihydrohomofolate and tetrahydrohomofolate (H 4HPteGlu) were synthesized and tested as inhibitors of folate-requiring enzymes. The following L. casei enzymes were examined: thymidylate synthase (TS), glycinamide ribonucleotide formyltransferase (GARFT), aminoimidazolecarboxamide ribonucleotide formyltransferase, serine hydroxymethyltransferase and dihydrofolate reductase. Polyglutamates of (6 R,S)-H 4HPteGlu are potent inhibitors of TS and GARFT. For example, the IC 50 values of (6 R,S)-H 4HPteGlu 6 are 0.7 μ m for TS and 0.3 μ m for GARFT. By contrast, the value for HPteGlu 6 is > 10 μ m for both TS and GARFT. Inhibition of TS and GARFT by (6 R,S)-H 4HPteGlu derivatives increases with polyglutamate chain length. For TS, the Glu 5 and Glu 6 derivatives of (6 R,S)-H 4HPteGlu are 20 and 30 times more potent than the monoglutamate, respectively. For GARFT, the Glu 2–6 derivatives are 2–3 times more potent than Glu 1. Inhibition of TS and GARFT by (6 R,S)-H 4HPteGlu polyglutamates is almost entirely due to the unnatural (6 R) diastereomer at C-6. Homofolate derivatives are only weak inhibitors of aminoimidazolecarboxamide ribonucleotide formyltransferase, serine hydroxymethyltransferase, and dihydrofolate reductase. We conclude that both TS and GARFT are potential targets of (6 R)-H 4HPteGlu polyglutamates.

Synthesis and antifolate activity of 5-methyl-5,10-dideaza analogues of aminopterin and folic acid and an alternative synthesis of 5,10-dideazatetrahydrofolic acid, a potent inhibitor of glycinamide ribonucleotide formyltransferase.

The title compounds were prepared in extensions of a general synthetic approach used earlier to prepare 5-alkyl-5-deaza analogues of classical antifolates. Wittig condensation of 2,4-diaminopyrido[2,3-d]pyrimidine-6-carboxaldehyde (2a) and its 5-methyl analogue 2b with [4-(methoxycarbonyl)benzylidene] triphenylphosphorane gave 9,10-ethenyl precursors 3a and 3b. Hydrogenation (DMF, ambient, 5% Pd/C) of the 9,10-ethenyl group of 3b followed by ester hydrolysis led to 4-[2-(2,4-diamino-5-methylpyrido[2,3-d]pyrimidin-6-yl)ethyl]ben zoi c acid (5), which was converted to 5-methyl-5,10-dideazaaminopterin (6) via coupling with dimethyl L-glutamate (mixed-anhydride method using i-BuOCOCl) followed by ester hydrolysis. Standard hydrolytic deamination of 6 gave 5-methyl-5,10-dideazafolic acid (7). Intermediates 3a and 3b were converted through concomitant deamination and ester hydrolysis to 8a and 8b. Peptide coupling of 8a,b (using (EtO)2POCN) with diesters of L-glutamic acid gave intermediate esters 9a and 9b. Hydrogenation of both the 9,10 double bond and the pyrido ring of 9a and 9b (MeOH-0.1 N HCl, 3.5 atm, Pt) was followed by ester hydrolysis to give 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (11a) and the 5-methyl analogue 11b. Biological evaluation of 6, 7, 11a, and 11b for inhibition of dihydrofolate reductase (DHFR) isolated from L1210 cells and for growth inhibition and transport characteristics toward L1210 cells revealed 6 to be less potent than methotrexate in the inhibition of DHFR and cell growth. Compounds 6, 11a, and 11b were transported into cells more efficiently than methotrexate. Growth inhibition IC50 values for 11a and 11b were 57 and 490 nM, respectively; the value for 11a is in good agreement with that previously reported (20-50 nM). In tests against other folate-utilizing enzymes, 11a and 11b were found to be inhibitors of glycinamide ribonucleotide formyltransferase (GAR formyltransferase) from one bacterial (Lactobacillus casei) and two mammalian (Manca and L1210) sources with 11a being decidedly more inhibitory than 11b. Neither 11a nor 11b inhibited aminoimidazolecarboxamide ribonucleotide formyltransferase. These results support reported evidence that 11a owes its observed antitumor activity to interference with the purine de novo pathway with the site of action being GAR formyltransferase.