Biosynthesis of Oxetanocin-A Includes a B12-Dependent Radical SAM Enzyme That Can Catalyze both Oxidative Ring Contraction and the Demethylation of SAM.

Oxetanocin-A is an antitumor, antiviral, and antibacterial nucleoside. It is biosynthesized via the oxidative ring contraction of a purine nucleoside co-opted from primary metabolism. This reaction is catalyzed by a B12-dependent radical S-adenosyl-l-methionine (SAM) enzyme, OxsB, and a phosphohydrolase, OxsA. Previous experiments showed that the product of the OxsB/OxsA-catalyzed reaction is an oxetane aldehyde produced alongside an uncharacterized byproduct. Experiments reported herein reveal that OxsB/OxsA complex formation is crucial for the ring contraction reaction and that reduction of the aldehyde intermediate is catalyzed by a nonspecific dehydrogenase from the general cellular pool. In addition, the byproduct is identified as a 1,3-thiazinane adduct between the aldehyde and l-homocysteine. While homocysteine was never included in the OxsB/OxsA assays, the data suggest that it can be generated from SAM via S-adenosyl-l-homocysteine (SAH). Further study revealed that conversion of SAM to SAH is facilitated by OxsB; however, the subsequent conversion of SAH to homocysteine is due to protein contaminants that co-purify with OxsA. Nevertheless, the observed demethylation of SAM to SAH suggests possible methyltransferase activity of OxsB, and substrate methylation was indeed detected in the OxsB-catalyzed reaction. This work is significant because it not only completes the description of the oxetanocin-A biosynthetic pathway but also suggests that OxsB may be capable of methyltransferase activity.

De novo transcriptome sequencing of Paecilomyces tenuipes revealed genes involved in adenosine biosynthesis.

The use of Paecilomyces tenuipes (P. tenuipes), a Chinese medicinal fungus in scientific research, is limited due to its low adenosine content. To improve adenosine production, the present study investigated the gene network of adenosine biosynthesis in P. tenuipes via transcriptome analysis. Mycelia of P. tenuipes cultured for 24 h (PT24), 102 h (PT102) and 196 h (PT192) were subjected to RNA sequencing. In total, 13,353 unigenes were obtained. Based on sequence similarity, 8,099 unigenes were annotated with known proteins. Of these 8,099 unigenes, 5,123 had functions assigned based on Gene Ontology terms while 4,158 were annotated based on the Eukaryotic Orthologous Groups database. Moreover, 1,272 unigenes were mapped to 281 Kyoto Encyclopedia of Genes and Genomes pathways. In addition, the differential gene expression of the three libraries was also performed. A total of 601, 1,658 and 628 differentially expressed genes (DEGs) were detected in PT24 vs. PT102, PT24 vs. PT192 and PT102 vs. PT192 groups, respectively. Reverse transcription­quantitative PCR was performed to analyze the expression levels of 14 DEGs putatively associated with adenosine biosynthesis in P. tenuipes. The results showed that two DEGs were closely associated with adenosine accumulation of P. tenuipes. The present study not only provides an improved understanding of the genetic information of P. tenuipes but also the findings can be used to aid research into P. tenuipes.

Cobalamin-dependent radical S-adenosyl-l-methionine enzymes in natural product biosynthesis.

Covering: 2011 to 2018 This highlight summarizes the investigation of cobalamin (Cbl)- and radical S-adenosyl-l-methionine (SAM)-dependent enzymes found in natural product biosynthesis to date and suggests some possibilities for the future. Though some mechanistic aspects are apparently shared, the overall diversity of this family's functions and abilities is significant and may be tailored to the specific substrate and/or reaction being catalyzed. A little over a year ago, the first crystal structure of a Cbl- and radical SAM-dependent enzyme was solved, providing the first insight into what may be the shared scaffolding of these enzymes.

Translational regulation by miR-301b upregulates AMP deaminase in diabetic hearts.

AMP deaminase (AMPD) plays a crucial role in adenine nucleotide metabolism. Recently we found that upregulated AMPD activity is associated with ATP depletion and contractile dysfunction under the condition of pressure overloading in the heart of a rat model of type 2 diabetes mellitus (T2DM), OLETF. Here we examined the mechanism of AMPD upregulation by T2DM. The protein level of 90-kDa full-length AMPD3 was increased in whole myocardial lysates by 55% in OLETF compared to those in LETO, a non-diabetic control. In contrast, the mRNA levels of AMPD3 in the myocardium were similar in OLETF and LETO. AMPD3 was comparably ubiquitinated in OLETF and LETO, and its degradation ex vivo was more sensitive to MG-132, a proteasome inhibitor, in OLETF than in LETO. MicroRNA array analysis revealed downregulation (>50%) of 57 microRNAs in OLETF compared to those in LETO, among which miR-301b was predicted to interact with the 3'UTR of AMPD3 mRNA. AMPD3 protein level was significantly increased by a miR-301b inhibitor and was decreased by a miR-301b mimetic in H9c2 cells. A luciferase reporter assay confirmed binding of miR-301b to the 3'UTR of AMPD3 mRNA. Transfection of neonatal rat cardiomyocytes with a miR-301b inhibitor increased 90-kDa AMPD3 and reduced ATP level. The results indicate that translational regulation by miR-301b mediates upregulated expression of cardiac AMPD3 protein in OLETF, which potentially reduces the adenine nucleotide pool at the time of increased work load. The miR-301b-AMPD3 axis may be a novel therapeutic target for intervening enegy metabolism in diabetic hearts.

C-C bond forming radical SAM enzymes involved in the construction of carbon skeletons of cofactors and natural products.

Covering: up to the end of 2017 C-C bond formations are frequently the key steps in cofactor and natural product biosynthesis. Historically, C-C bond formations were thought to proceed by two electron mechanisms, represented by Claisen condensation in fatty acids and polyketide biosynthesis. These types of mechanisms require activated substrates to create a nucleophile and an electrophile. More recently, increasing number of C-C bond formations catalyzed by radical SAM enzymes are being identified. These free radical mediated reactions can proceed between almost any sp3 and sp2 carbon centers, allowing introduction of C-C bonds at unconventional positions in metabolites. Therefore, free radical mediated C-C bond formations are frequently found in the construction of structurally unique and complex metabolites. This review discusses our current understanding of the functions and mechanisms of C-C bond forming radical SAM enzymes and highlights their important roles in the biosynthesis of structurally complex, naturally occurring organic molecules. Mechanistic consideration of C-C bond formation by radical SAM enzymes identifies the significance of three key mechanistic factors: radical initiation, acceptor substrate activation and radical quenching. Understanding the functions and mechanisms of these characteristic enzymes will be important not only in promoting our understanding of radical SAM enzymes, but also for understanding natural product and cofactor biosynthesis.

A B12-dependent radical SAM enzyme involved in oxetanocin A biosynthesis.

Oxetanocin A (OXT-A) is a potent antitumour, antiviral and antibacterial compound. Biosynthesis of OXT-A has been linked to a plasmid-borne Bacillus megaterium gene cluster that contains four genes: oxsA, oxsB, oxrA and oxrB. Here we show that both the oxsA and oxsB genes are required for the production of OXT-A. Biochemical analysis of the encoded proteins, a cobalamin (Cbl)-dependent S-adenosylmethionine (AdoMet) radical enzyme, OxsB, and an HD-domain phosphohydrolase, OxsA, reveals that OXT-A is derived from a 2'-deoxyadenosine phosphate in an OxsB-catalysed ring contraction reaction initiated by hydrogen atom abstraction from C2'. Hence, OxsB represents the first biochemically characterized non-methylating Cbl-dependent AdoMet radical enzyme. X-ray analysis of OxsB reveals the fold of a Cbl-dependent AdoMet radical enzyme, a family of enzymes with an estimated 7,000 members. Overall, this work provides a framework for understanding the interplay of AdoMet and Cbl cofactors and expands the catalytic repertoire of Cbl-dependent AdoMet radical enzymes.

Butyrate influences intracellular levels of adenine and adenine derivatives in the fungus Penicillium restrictum.

Butyrate, a small fatty acid, has an important role in the colon of ruminants and mammalians including the inhibition of inflammation and the regulation of cell proliferation. There is also growing evidence that butyrate is influencing the histone structure in mammalian cells by inhibition of histone deacetylation. Butyrate shows furthermore an antimicrobial activity against fungi, yeast and bacteria, which is linked to its toxicity at a high concentration. In fungi there are indications that butyrate induces the production of secondary metabolites potentially via inhibition of histone deacetylases. However, information about the influence of butyrate on growth, primary metabolite production and metabolism, besides lipid catabolism, in fungi is scarce. We have identified the filamentous fungus Penicillium (P.) restrictum as a susceptible target for butyrate treatment in an antimicrobial activity screen. The antimicrobial activity was detected only in the mycelium of the butyrate treated culture. We investigated the effect of butyrate ranging from low (0.001mM) to high (30mM), potentially toxic, concentrations on biomass and antimicrobial activity. Butyrate at high concentrations (3 and 30mM) significantly reduced the fungal biomass. In contrast P. restrictum treated with 0.03mM of butyrate showed the highest antimicrobial activity. We isolated three antimicrobial active compounds, active against Staphylococcus aureus, from P. restrictum cellular extracts treated with butyrate: adenine, its derivate hypoxanthine and the nucleoside derivate adenosine. Production of all three compounds was increased at low butyrate concentrations. Furthermore we found that butyrate influences the intracellular level of the adenine nucleoside derivate cAMP, an important signalling molecule in fungi and various organisms. In conclusion butyrate treatment increases the intracellular levels of adenine and its respective derivatives.

An HD domain phosphohydrolase active site tailored for oxetanocin-A biosynthesis.

HD domain phosphohydrolase enzymes are characterized by a conserved set of histidine and aspartate residues that coordinate an active site metallocenter. Despite the important roles these enzymes play in nucleotide metabolism and signal transduction, few have been both biochemically and structurally characterized. Here, we present X-ray crystal structures and biochemical characterization of the Bacillus megaterium HD domain phosphohydrolase OxsA, involved in the biosynthesis of the antitumor, antiviral, and antibacterial compound oxetanocin-A. These studies reveal a previously uncharacterized reaction for this family; OxsA catalyzes the conversion of a triphosphorylated compound into a nucleoside, releasing one molecule of inorganic phosphate at a time. Remarkably, this functionality is a result of the OxsA active site, which based on structural and kinetic analyses has been tailored to bind the small, four-membered ring of oxetanocin-A over larger substrates. Furthermore, our OxsA structures show an active site that switches from a dinuclear to a mononuclear metal center as phosphates are eliminated from substrate.

High-spin ferric ions in Saccharomyces cerevisiae vacuoles are reduced to the ferrous state during adenine-precursor detoxification.

The majority of Fe in Fe-replete yeast cells is located in vacuoles. These acidic organelles store Fe for use under Fe-deficient conditions and they sequester it from other parts of the cell to avoid Fe-associated toxicity. Vacuolar Fe is predominantly in the form of one or more magnetically isolated nonheme high-spin (NHHS) Fe(III) complexes with polyphosphate-related ligands. Some Fe(III) oxyhydroxide nanoparticles may also be present in these organelles, perhaps in equilibrium with the NHHS Fe(III). Little is known regarding the chemical properties of vacuolar Fe. When grown on adenine-deficient medium (A↓), ADE2Δ strains of yeast such as W303 produce a toxic intermediate in the adenine biosynthetic pathway. This intermediate is conjugated with glutathione and shuttled into the vacuole for detoxification. The iron content of A↓ W303 cells was determined by Mössbauer and EPR spectroscopies. As they transitioned from exponential growth to stationary state, A↓ cells (supplemented with 40 µM Fe(III) citrate) accumulated two major NHHS Fe(II) species as the vacuolar NHHS Fe(III) species declined. This is evidence that vacuoles in A↓ cells are more reducing than those in adenine-sufficient cells. A↓ cells suffered less oxidative stress despite the abundance of NHHS Fe(II) complexes; such species typically promote Fenton chemistry. Most Fe in cells grown for 5 days with extra yeast-nitrogen-base, amino acids and bases in minimal medium was HS Fe(III) with insignificant amounts of nanoparticles. The vacuoles of these cells might be more acidic than normal and can accommodate high concentrations of HS Fe(III) species. Glucose levels and rapamycin (affecting the TOR system) affected cellular Fe content. This study illustrates the sensitivity of cellular Fe to changes in metabolism, redox state and pH. Such effects broaden our understanding of how Fe and overall cellular metabolism are integrated.

Mechanism of N6-threonylcarbamoyladenonsine (t(6)A) biosynthesis: isolation and characterization of the intermediate threonylcarbamoyl-AMP.

Genetic and biochemical studies have recently implicated four proteins required in bacteria for the biosynthesis of the universal tRNA modified base N6-threonylcarbamoyl adenosine (t(6)A). In this work, t(6)A biosynthesis in Bacillus subtilis has been reconstituted in vitro and found to indeed require the four proteins YwlC (TsaC), YdiB (TsaE), YdiC (TsaB) and YdiE (TsaD). YwlC was found to catalyze the conversion of L-threonine, bicarbonate/CO(2) and ATP to give the intermediate L-threonylcarbamoyl-AMP (TC-AMP) and pyrophosphate as products. TC-AMP was isolated by HPLC and characterized by mass spectrometry and (1)H NMR. NMR analysis showed that TC-AMP decomposes to give AMP and a nearly equimolar mixture of L-threonine and 5-methyl-2-oxazolidinone-4-carboxylate as final products. Under physiological conditions (pH 7.5, 37 °C, 2 mM MgCl(2)), the half-life of TC-AMP was measured to be 3.5 min. Both YwlC (in the presence of pyrophosphatase) and its Escherichia coli homologue YrdC catalyze the formation of TC-AMP while producing only a small molar fraction of AMP. This suggests that CO(2) and not an activated form of bicarbonate is the true substrate for these enzymes. In the presence of pyrophosphate, both enzymes catalyze clean conversion of TC-AMP back to ATP. Purified TC-AMP is efficiently processed to t(6)A by the YdiBCE proteins in the presence of tRNA substrates. This reaction is ATP independent in vitro, despite the known ATPase activity of YdiB. The estimated rate of conversion of TC-AMP by YdiBCE to t(6)A is somewhat lower than the initial rate from L-threonine, bicarbonate and ATP, which together with the stability data, is consistent with previous studies that suggest channeling of this intermediate.

A fundamental regulatory role of formate on thuringiensin production by resting cell of Bacillus thuringiensis YBT-032.

In this work, a fundamental regulatory role of formate on thuringiensin production by resting cell of Bacillus thuringiensis YBT-032 was investigated. Nicotinamide adenine dinucleotide (NADH) production and formate dehydrogenase activity increased with formate addition from 0.5 to 2.0 g/L, respectively. However, with the formate addition of 1.5 g/L, the activities of pyruvate kinase and glucose 6-phosphate dehydrogenase reached a peak and increased by 316 and 150% relative to those of the control, respectively. In addition, intracellular production of pyruvate, aspartate, citrate and adenine were significantly enhanced by 75, 66, 32 and 78% as well. An improvement (90%) of thuringiensin production was also successfully obtained. Interestingly to point out, thuringiensin yield was closely correlative with adenine production, and the linear relationship was also observed. The results suggest that appropriate formate addition did act as a modulator and facilitate carbon flux in glycolysis and pentose phosphate pathway to synthesize adenine and thuringiensin via intracellular NADH availability.

A survey of all 11 ABC transporters in fission yeast: two novel ABC transporters are required for red pigment accumulation in a Schizosaccharomyces pombe adenine biosynthetic mutant.

ATP-binding cassette (ABC) proteins transport a wide variety of substrates, including sugars, amino acids, metal ions, lipids, peptides and proteins, across membranes, and most ABC proteins contain transmembrane domains (ABC transporters). Sequencing of the Schizosaccharomyces pombe genome has allowed identification of all genes encoding ABC transporters in fission yeast. To date, six such genes have been characterized, and an additional five genes encoding ABC transporters were identified from the genome sequence. In an attempt to characterize all of the ABC transporters in fission yeast, all 11 genes were disrupted. While all the genes were found to be dispensable for cell viability, some disruptants lacked apparent phenotypes. GFP-tagged ABC transporters were localized to membranes as follows: plasma membrane (2), vacuolar membrane (4), mitochondrial membrane (2), endoplasmic reticulum membrane (2), and endosome and Golgi membranes (1). Two Cluster II. 1 proteins, Abc2p (SPAC3F10.11c) and Abc4p (SPAC30.04c), were found to be localized to vacuolar membranes, and to be responsible for accumulation of a characteristic red pigment in the vacuole of an adenine biosynthetic mutant. The doubly disrupted mutant abc2 Delta abc4 Delta exhibited drug sensitivity, and a decreased accumulation of monochlorobimane, suggesting that both of the proteins encoded by these genes are involved in detoxification of xenobiotics, and vacuolar sequestration of glutathione S-conjugates.

Immobilized Escherichia coli BL21 as a catalyst for the synthesis of adenine and hypoxanthine nucleosides.

Different supports, such as alginate, agar, agarose, and polyacrylamide, were used to immobilize Escherichia coli BL 21 by entrapment techniques. The transglycosylation reaction involved in the synthesis of adenosine from uridine and adenine was chosen as a model system to study the characteristics of these biocatalysts. Whole cells immobilized on agarose proved to be optimal and could be used up to 30 times without significant loss of activity. This biocatalyst was further employed to test its ability in the synthesis of other adenine and hypoxanthine nucleosides. Ribo-, 2'-deoxyribo-, and arabinonucleosides could be prepared in high yields starting from the corresponding pyrimidine nucleosides and purine bases. Similar product yields were obtained with both free and immobilized cells, though, in the latter case, a longer reaction time was necessary.

Detection and quantification of 1,N6-ethenoadenine in human urine by stable isotope dilution capillary gas chromatography/negative ion chemical ionization/mass spectrometry.

1,N(6)-Ethenoadenine (epsilonAde) is a promutagenic lesion detected in tissue DNA; it has been shown that epsilonAde can be repaired by human DNA glycosylases, and it is expected to be excreted in urine. In this paper, we present for the first time detection and accurate quantification of epsilonAde in human urine samples by a highly sensitive and specific stable isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometric assay (GC/NICI/MS). Analysis by GC/NICI/MS includes adduct enrichment by a solid phase extraction column, followed by electrophore labeling and postderivatization cleanup. Using selective ion monitoring mode, the assay allows quantification of 0.5 pg of epsilonAde in as little as 0.1 mL of the urine sample, which is equivalent to corresponding concentration quantification limit of 31 pM. Using this assay, concentrations of epsilonAde in the 24 h urine samples of 23 healthy individuals were determined, which ranged from 0 to 124 pg/mL. After we adjusted for creatinine, a statistically significant correlation was found between epsilonAde excretion and cigarette smoking in males (p = 0.03). Thus, this stable isotope dilution GC/NICI/MS assay offers a sensitive and accurate quantification of urinary epsilonAde as a potential biomarker for oxidative damage of DNA and repair.

Development of a whole cell assay to measure methotrexate-induced inhibition of thymidylate synthase and de novo purine synthesis in leukaemia cells.

The cellular pharmacology of methotrexate (MTX) is complex, involving the inhibition of both de novo thymidylate and purine biosynthesis. Measurement of MTX-induced inhibition of de novo thymidylate and purine biosynthesis may allow optimisation of MTX therapy, and the aim of this study was to develop an assay to measure the activity of both pathways in the same cell sample, and so determine the effects of MTX treatment. In situ thymidylate synthase (EC 2.1.1.45) activity was measured by the release of 3H2O from [5'-3H]deoxyuridine and de novo purine synthesis by the incorporation of [14C]formate into adenine and guanine. Incubation of human leukaemia CCRF-CEM cells for 22 hr with 50 nM MTX resulted in approximately 90% inhibition of in situ thymidylate synthase activity, relative to control untreated cells, and after exposure to 1000 nM MTX activity could not be detected. In contrast, de novo purine synthesis, measured in the same sample, was not inhibited by exposure to 50 nM MTX, although activity was again completely abolished by exposure to 1000 nM MTX. To demonstrate the utility of the assay, lymphoblasts isolated from a child with acute lymphoblastic leukaemia (ALL) were also incubated for 22 hr with 1000 nM MTX. Both in situ thymidylate synthase activity and de novo purine synthesis were significantly inhibited, by 70% and 60% respectively, relative to the activity in untreated cells.

Methyl donor for 1-methyladenine biosynthesis in starfish ovarian follicle cells.

1-Methyladenine (1-MeAde), the oocyte maturation-inducing substance of starfish, is produced by ovarian follicle cells upon stimulation with a gonad-stimulating substance (GSS) released from radial nerves. It has been reported that a process of methylation is involved in GSS-induced 1-MeAde production by starfish ovarian follicle cells. The present study sought to identify a possible methyl donor for 1-MeAde biosynthesis in follicle cells of the starfish Asterina pectinifera. When isolated follicle cells were incubated with [methyl-(14)C]methionine (Met), there was an increase in the level of radiolabeled S-adenosylmethionine (SAM). After further incubation with GSS, the [methyl-(14)C]SAM level decreased, concomitant with a marked increase in radiolabeled 1-MeAde production. The amount of [methyl-(14)C]SAM consumed under the influence of GSS was similar to the amount of [methyl-(14)C]1-MeAde produced. Therefore, it is concluded that SAM is a methyl donor for 1-MeAde biosynthesis in starfish ovarian follicle cells. On the other hand, it is likely that the purine molecule of 1-MeAde is not derived from SAM but from ATP. 3-Isobutyl-1-methylxanthine, a potent inhibitor of cyclic AMP phosphodiesterase, also caused a reduction in the level of radiolabeled SAM following 1-MeAde production. This suggests that the methylation process of 1-MeAde biosynthesis in starfish ovarian follicle cells upon stimulation with GSS is mediated by a second messenger, cyclic AMP.

1-Methyladenine production from ATP by starfish ovarian follicle cells.

1-Methyladenine (1-MeAde), the oocyte maturation-inducing substance in starfish, is produced by ovarian follicle cells upon stimulation with a gonad-stimulating substance (GSS) released from the radial nerves. We have shown previously that GSS causes a reduction in the intracellular levels of ATP coincident with 1-MeAde production. The present study examined whether the adenine molecule of 1-MeAde is directly derived from ATP. When isolated follicle cells from the starfish Asterina pectinifera were preloaded with [U-14C]adenine or [U-14C]adenosine, there was an increase in the intracellular levels of radiolabeled adenine nucleotides, particularly ATP. Following further incubation with GSS, the intracellular levels of radiolabeled ATP decreased, concomitant with a marked increase in the levels of [14C]1-MeAde in the medium. The amount of ATP consumed under the influence of GSS was similar to the amount of 1-MeAde produced. However, there was no change in the levels of ADP and AMP regardless of the presence or absence of GSS. These findings strongly suggest that 1-MeAde is synthesized from ATP as a substrate in follicle cells under the influence of GSS. Furthermore, using [methyl-3H]methionine, the methyl group of 1-MeAde was found to be derived from methionine. Thus GSS appears to stimulate the synthesis of 1-MeAde from ATP via the methylation process in starfish ovarian follicle cells.

The prebiotic synthesis of modified purines and their potential role in the RNA world.

Modified purines are found in all organisms in the tRNA, rRNA, and even DNA, raising the possibility of an early role for these compounds in the evolution of life. These include N6-methyladenine, 1-methyladenine, N6,N6-dimethyladenine, 1-methylhypoxanthine, 1-methylguanine, and N2-methylguanine. We find that these bases as well as a number of nonbiological modified purines can be synthesized from adenine and guanine by the simple reaction of an amine or an amino group with adenine and guanine under the concentrated conditions of the drying-lagoon or drying-beach model of prebiotic synthesis with yields as high as 50%. These compounds are therefore as prebiotic as adenine and guanine and could have played an important role in the RNA world by providing additional functional groups in ribozymes, especially for the construction of hydrophobic binding pockets.