Biosynthesis of the 3-benzylchroman-4-one eucomin in Eucomis bicolor

Feeding experiments have demonstrated the specific incorporation of radioactivity from dl-phenylalanine-[1-¹⁴C], l-phenylalanine-[U-¹⁴C], sodium acetate-[2-¹⁴C] and l-methionine-[methyl-¹⁴C] into the 3-benzylchroman-4-one eucomin in Eucomis bicolor. The labelling patterns indicate that eucomin is biosynthesized by the addition of a carbon atom derived from methionine onto a C₁₅ chalcone-type skeleton. Radioactivity from 2′,4′,4-trihydroxy-6′-methoxychalcone-[methyl-¹⁴C] and 2′,4′-dihydroxy-4,6′-dimethoxychalcone-[6′-methyl-¹⁴C] was incorporated into eucomin, the latter compound being the better precursor, demonstrating the feasibility that 2′-methoxychalcones are biosynthetic precursors of the “homoisoflavonoids”. Possible biosynthetic relationships in this class of compounds are discussed.     

Change in the levels of adenine-related compounds in starfish ovarian follicle cells following treatment with gonad-stimulating substance

The resumption of meiosis in starfish oocytes is induced by 1-methyladenine (1-MeA), which is produced by ovarian foilicle cells under the influence of a gonad-stimulating substance (GSS). It has been reported that the 1-MeA produced is newly synthesized via a process of methylation, rather than being pre-stored within follicle cells or a breakdown product of some 1-MeA-containing substance. The present study examined a possible substrate for 1-MeA biosynthesis stored in follicle cells of the starfish Asterina pectinifera . Analyses using high-performance liquid chromatography indicated a large source of ATP among the adenine-related compounds in these follicle cells. When follicle cells were incubated in seawater in the presence of GSS, 1-MeA production was stimulated significantly. GSS also caused a reduction in intracellular levels of ATP. There was no change in the levels of either ADP or AMP. The amount of ATP consumed under the influence of GSS was similar to the amount of 1-MeA produced. Methionine and selenomethionine enhanced both 1-MeA production and ATP consumption by GSS in follicle cells. In contrast, ethionine and selenoethionine, competitive inhibitors of methionine, inhibited these processes. These results suggest that ATP is a possible substrate in the biosynthesis of 1-MeA by starfish ovarian follicle cells.     

Acyl-chain remodeling of dioctanoyl-phosphatidylcholine in Saccharomyces cerevisiae mutant defective in de novo and salvage phosphatidylcholine synthesis

A yeast strain, in which endogenous phosphatidylcholine (PC) synthesis is controllable, was constructed by the replacement of the promoter of PCT1, encoding CTP:phosphocholine cytidylyltransferase, with GAL1 promoter in a double deletion mutant of PEM1 and PEM2, encoding phosphatidylethanolamine methyltransferase and phospholipid methyltransferase, respectively. This mutant did not grow in the glucose-containing medium, but the addition of dioctanoyl-phosphatidylcholine (diC8PC) supported its growth. Analyses of the metabolism of ¹³C-labeled diC8PC ((methyl-¹³C)₃-diC8PC) in this strain using electrospray ionization tandem mass spectrometry revealed that it was converted to PC species containing acyl residues of 16 or 18 carbons at both sn-1 and sn-2 positions. In addition, both acyl residues of (methyl-¹³C)₃-diC8PC were replaced with 16:1 acyl chains in the in vitro reaction using the yeast cell extract in the presence of palmitoleoyl-CoA. These results indicate that PC containing short acyl residues was remodeled to those with acyl chains of physiological length in yeast.     

1-Methyladenine inhibits adenylate cyclase in starfish oocytes

Summary

The effect of 1-methyladenine (1-MeA) on adenylate cyclase (AC) basal activity and on preliminary stimulated AC activity was investigated in oocyte membrane preparations of the starfish Aphelasterias japonica. 1-MeA inhibited the membrane-bound AC activity both after its addition to intact oocytes and in cell-free experiments. GTP did not affect AC activity but it intensified the inhibitory effect of 1-MeA on AC activity. Sodium fluoride (F″) stimulated the oocyte AC (8 fold), while 1-MeA significantly reduced F″-stimulated activity. Manganese (MnCl₂, 5mM) stimulated AC (150 fold), but 1-MeA did not reduce Mn²⁺-stimulated activity. However, Mn²⁺-stimulated AC activity was inhibited by 1-MeA in the presence of MgCl₂. Forskolin stimulated AC activity (7 fold) and 1-MeA had no effect on AC. Thus, the inhibitory effect of 1-MeA on stimulated AC activity is displayed only after stimulation of the regulatory AC subunit. We suggest that 1-MeA inhibits the oocyte AC acting via inhibitory regulatory Gi protein of AC.     

Biosynthesis of internally branched monomethylalkanes in the cockroach Periplaneta fuliginosa.

Sodium [1-¹⁴C]acetate, sodium [1-¹⁴C]propionate, sodium [2-¹⁴C]propionate, sodium [3-¹⁴C]propionate and sodium [methyl-¹⁴C]methylmalonate were readily incorporated into the cuticular hydrocarbons of nymphal stages of the cockroach Periplaneta fuliginosa both in vivo and in vitro, whereas no incorporation of [methyl-¹⁴C]methionine was observed. The alkanes of the nymphal stages of this insect are 25+% n-alkanes, 14% 3-methylalkanes, and 59+% internally branched monomethylalkanes, principally 13-methylpentacosane. Sodium [1-¹⁴C]acetate was incorporated into each class of alkane at about its percentage composition. In contrast, labeled sodium propionate and sodium methylmalonate were preferentially incorporated into the branched fractions. Radio-gas-liquid chromatography showed that sodium [1-¹⁴C]propionate was incorporated almost exclusively into 3-methyltricosane and 13-methylpentacosane, whereas sodium [1-¹⁴C]acetate was incorporated into each glc peak at about its percentage composition. These data suggest that propionate, incorporated during chain elongation, serves as the branching methyl group donor for both the 3-methyl and the internally branched monomethylalkanes in insects. The location of hydrocarbon synthesis in P. fuliginosa was studied using an in vitro tissue slice system. Excised cuticle slices, with adhering fat body tissue removed, gave good incorporation of labeled substrates into the hydrocarbon fraction. No hydrocarbon synthesis was observed in fat body preparations.     

DNA polymerase λ promotes error-free replication through Watson–Crick impairing N1-methyl-deoxyadenosine adduct in conjunction with DNA polymerase ζ

In a previous study, we showed that replication through the N1-methyl-deoxyadenosine (1-MeA) adduct in human cells is mediated via three different Polι/Polθ, Polη, and Polζ-dependent pathways. Based on biochemical studies with these Pols, in the Polι/Polθ pathway, we inferred a role for Polι in the insertion of a nucleotide (nt) opposite 1-MeA and of Polθ in extension of synthesis from the inserted nt; in the Polη pathway, we inferred that this Pol alone would replicate through 1-MeA; in the Polζ pathway, however, the Pol required for inserting an nt opposite 1-MeA had remained unidentified. In this study, we provide biochemical and genetic evidence for a role for Polλ in inserting the correct nt T opposite 1-MeA, from which Polζ would extend synthesis. The high proficiency of purified Polλ for inserting a T opposite 1-MeA implicates a role for Polλ—which normally uses W-C base pairing for DNA synthesis—in accommodating 1-MeA in a syn confirmation and forming a Hoogsteen base pair with T. The potential of Polλ to replicate through DNA lesions by Hoogsteen base pairing adds another novel aspect to Polλ’s role in translesion synthesis in addition to its role as a scaffolding component of Polζ. We discuss how the action mechanisms of Polλ and Polζ could be restrained to inserting a T opposite 1-MeA and extending synthesis thereafter, respectively.     

Biosynthesis of (−)-ent-kaurenoic acid in Smallanthus sonchifolius and its effect against microbial biofilms

The biosynthetic pathway of (–)-ent-kaurenoic acid (1) was investigated by incorporation of 1-d-¹³C-glucose in Smallanthus sonchifolius (Asteraceae) plantlets. The ¹³C-enrichment pattern indicated that methylerythritol-4-phosphate (MEP) pathway is the biosynthetic pathway involved in diterpenoid biosynthesis. Our studies in S. sonchifolius reinforce that the biosynthesis of different classes of terpenes should not be compartmentalized into cytosol and plastids. Additionally, (–)-ent-kaurenoic acid showed antimicrobial activity against Staphylococcus aureus biofilm.     

Genetic Control of Replication through N1-methyladenine in Human Cells

N1-methyl adenine (1-MeA) is formed in DNA by reaction with alkylating agents and naturally occurring methyl halides. The 1-MeA lesion impairs Watson-Crick base pairing and blocks normal DNA replication. Here we identify the translesion synthesis (TLS) DNA polymerases (Pols) required for replicating through 1-MeA in human cells and show that TLS through this lesion is mediated via three different pathways in which Pols ι and θ function in one pathway and Pols η and ζ, respectively, function in the other two pathways. Our biochemical studies indicate that in the Polι/Polθ pathway, Polι would carry out nucleotide insertion opposite 1-MeA from which Polθ would extend synthesis. In the Polη pathway, this Pol alone would function at both the nucleotide insertion and extension steps of TLS, and in the third pathway, Polζ would extend from the nucleotide inserted opposite 1-MeA by an as yet unidentified Pol. Whereas by pushing 1-MeA into the syn conformation and by forming Hoogsteen base pair with the T residue, Polι would carry out TLS opposite 1-MeA, the ability of Polη to replicate through 1-MeA suggests that despite its need for Watson-Crick hydrogen bonding, Polη can stabilize the adduct in its active site. Remarkably, even though Pols η and ι are quite error-prone at inserting nucleotides opposite 1-MeA, TLS opposite this lesion in human cells occurs in a highly error-free fashion. This suggests that the in vivo fidelity of TLS Pols is regulated by factors such as post-translational modifications, protein-protein interactions, and possibly others.     

Biosynthesis of polar steroids from the Far Eastern starfish Patiria (=Asterina) pectinifera. Cholesterol and cholesterol sulfate are converted into polyhydroxylated sterols and monoglycoside asterosaponin P1 in feeding experiments

For the first time, it is experimentally established that the dietary cholesterol and cholesterol sulfate are biosynthetic precursors of polyhydroxysteroids and related low molecular weight glycosides in starfishes. These deuterium labeled precursors were converted into partly deuterated 5α-cholestane-3β,6α,7α,8,15α,16β,26-heptaol, 5α-cholestane-3β,4β,6α,7α,8,15β,16β,26-octaol, and steroid monoside asterosaponin P₁ in result of feeding experiments on the Far Eastern starfish Patiria (=Asterina) pectinifera. The incorporations of deuterium were established by MS and NMR spectroscopy. Scheme of the first stages of biosynthesis of polar steroids in these animals was suggested on the basis of inclusion of three from six deuterium atoms and determination of their positions in biosynthetic products, when [2,2,3,4,4,6-²H₆]cholesterol 3-sulfate was used as precursor. It was also shown that labeled cholesterol is transformed into Δ⁷-cholesterol (lathosterol) in digestive organs and gonads of the starfish.     

A radioactive assay for guanidoacetate methyltransferase.

A radioactive assay for guanidoacetate methyltransferase is described. It is based on a separation by an AG 50 (NH₄⁺) column, on which [methyl-¹⁴C]Adenosylmethionine is adsorbed, in contrast to [methyl-¹⁴C]creatine which can be collected in the effluent fraction. Guanidoacetate methyltransferase is sensitive to inhibition by adenosylhomocysteine and 3-deazaadenosylhomocysteine.     

Biosynthesis of N-methyl-l-glucosamine from d-glucose by Streptomyces griseus

Biosynthesis of $\text{N-}\text{methyl}\text{-}\text{l}\text{-}\text{glucosamine}$ moiety of streptomycin from d-glucose by Streptomyces griseus was studied. A mixture of d-[1-¹⁴C]glucose and d-[6-³H]glucose was given to the culture of S. griseus. The ³H/¹⁴C ratio found in $\text{N-}\text{methyl}\text{-}\text{d}\text{-}\text{glucosamine}$ further supports a mechanism that the conversion of d-glucose to l-hexose is carried out without scission of carbon skeleton. When d-[1-¹⁴C]glucose and d-[3-³H]glucose were used, the fall of ³H/¹⁴C ratio in $\text{N-}\text{methyl}\text{-}\text{l}\text{-}\text{glucosamine}$ showed that the hydrogen atom at C-3 plays a rôle in such a transformation.     

Biosynthesis of andrographolide in Andrographis paniculata

Andrographolide, a diterpene lactone, is isolated from Andrographis paniculata which is well known for its medicinal properties. The biosynthetic route to andrographolide was studied using [1-¹³C]acetate, [2-¹³C]acetate and [1,6-¹³C₂]glucose. The peak enrichment of eight carbon atoms in the ¹³C NMR spectra of andrographolide suggested that deoxyxylulose pathway (DXP) is the major biosynthetic pathway to this diterpene.The contribution of the mevalonic acid pathway (MVA) is indicated by the observed ¹³C-labeling pattern, and because the labeling patterns indicate a simultaneous contribution of both methyl erythritol phosphate (MEP) and MVA pathways it can be deduced that cross-talk occurs between plastids and cytoplasm.     

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‐¹⁴C]methionine (Met), there was an increase in the level of radiolabeled S‐adenosylmethionine (SAM). After further incubation with GSS, the [methyl‐¹⁴C]SAM level decreased, concomitant with a marked increase in radiolabeled 1‐MeAde production. The amount of [methyl‐¹⁴C]SAM consumed under the influence of GSS was similar to the amount of [methyl‐¹⁴C]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. Mol. Reprod. Dev. 54:63–68, 1999. © 1999 Wiley‐Liss, Inc.     

Role of Central Metabolism in the Osmoadaptation of the Halophilic Bacterium Chromohalobacter salexigens

Bacterial osmoadaptation involves the cytoplasmic accumulation of compatible solutes to counteract extracellular osmolarity. The halophilic and highly halotolerant bacterium Chromohalobacter salexigens is able to grow up to 3 m NaCl in a minimal medium due to the de novo synthesis of ectoines. This is an osmoregulated pathway that burdens central metabolic routes by quantitatively drawing off TCA cycle intermediaries. Consequently, metabolism in C. salexigens has adapted to support this biosynthetic route. Metabolism of C. salexigens is more efficient at high salinity than at low salinity, as reflected by lower glucose consumption, lower metabolite overflow, and higher biomass yield. At low salinity, by-products (mainly gluconate, pyruvate, and acetate) accumulate extracellularly. Using [1-¹³C]-, [2-¹³C]-, [6-¹³C]-, and [U-¹³C₆]glucose as carbon sources, we were able to determine the main central metabolic pathways involved in ectoines biosynthesis from glucose. C. salexigens uses the Entner-Doudoroff pathway rather than the standard glycolytic pathway for glucose catabolism, and anaplerotic activity is high to replenish the TCA cycle with the intermediaries withdrawn for ectoines biosynthesis. Metabolic flux ratios at low and high salinity were similar, revealing a certain metabolic rigidity, probably due to its specialization to support high biosynthetic fluxes and partially explaining why metabolic yields are so highly affected by salinity. This work represents an important contribution to the elucidation of specific metabolic adaptations in compatible solute-accumulating halophilic bacteria.     

A novel link between the biosynthesis of aromatic amino acids and transfer RNA modification in Escherichia coli

A transposon-induced mutation in Escherichia coli resulted in a lack of two modified nucleosides in the transfer ribonucleic acid. These nucleosides were identified as uridine-5-oxyacetic acid (cmo⁵U)² and its methylester, mcmo⁵U. Both became radioactively labelled using [methyl-¹⁴C]methionine as methyl donor when wild-type cells were grown in a defined rich medium. We believe that both nucleosides have hydroxyuridine as a common precursor, which should be methylated in the first modification step. However, in our system in vitro the tRNA from the mutant was not a methyl group acceptor, indicating that the step affected in the mutant occurs before the methylation step. Thus, the most likely biosynthetic pathway is: formation of (1) hydroxyuridine, (2) methoxyuridine. (3) cmo⁵U and, in some cases, (4) mcmo⁵U. The mutant had also become Aro^?, i.e. it required aromatic amino acids for growth. Genetic analysis revealed that the transposon Tn5 had been integrated close to or within the aroD gene, the gene product of which participates in the synthesis of shikimic acid. The common pathway of the biosynthesis of aromatic amino acids includes the genes aroB, D, E, A and C in that order, and any mutant defective in any of these genes lacked cmo⁵U and mcmo⁵U in their tRNA. When shikimic acid was included in the defined rich medium used, the Tn5-induced mutant regained the normal level of cmo⁵U and mcmo⁵U while an aroC mutant (distal to shikimic acid but prior to chorismic acid) did not. The rich medium used contained, besides the aromatic amino acids, all the precursors for the synthesis of folate, ubiquinone and enterochelin. Thus, chorismic acid itself or a metabolite of it in the synthetic pathway to vitamin K₂ or in an unknown pathway must play a pivotal role in this specific modification of the tRNA. These results reveal a novel link between the biosynthesis of amino acids and modification of tRNA.     

Pterocarpan biosynthesis: Chalone and isoflavone precursors of demethylhomopterocarpin and maackiain in Trifolium pratense

Feeding experiments with dl-phenylalanine-[1-¹⁴C] have demonstrated the de novo synthesis of the pterocarpan phytoalexins demethylhomopterocarpin and maackiain in CuCl₂-treated Trifolium pratense L. seedlings. 2′,4′,4-Trihydroxychalcone-[carbonyl-¹⁴C] and 7-hydroxy-4′-methoxyisoflavone-[methyl-¹⁴C] (formononetin) were readily incorporated into demethylhomopterocarpin and maackiain, but 2′,4′-dihydroxy-4-methoxychalcone-[carbonyl-¹⁴C] and 7,4′-dihydroxyisoflavone-[T] (daidzein) proved inefficient precursors. The trihydroxychalcone was also an excellent precursor of formononetin in T. pratense, but the trihydroxymethoxychalcone and daidzen were poorly incorporated. These observations offer further evidence that methylation may be an associated part of the mechanism for aryl migration in the biosynthesis of formononetin.     

Metabolism of One-Carbon Compounds by the Ruminal Acetogen Syntrophococcus sucromutans

Syntrophococcus sucromutans is the predominant species capable of O demethylation of methoxylated lignin monoaromatic derivatives in the rumen. The enzymatic characterization of this acetogen indicated that it uses the acetyl coenzyme A (Wood) pathway. Cell extracts possess all the enzymes of the tetrahydrofolate pathway, as well as carbon monoxide dehydrogenase, at levels similar to those of other acetogens using this pathway. However, formate dehydrogenase could not be detected in cell extracts, whether formate or a methoxyaromatic was used as electron acceptor for growth of the cells on cellobiose. Labeled bicarbonate, formate, [1-¹⁴C] pyruvate, and chemically synthesized O-[methyl-¹⁴C]vanillate were used to further investigate the catabolism of one-carbon (C₁) compounds by using washed-cell preparations. The results were consistent with little or no contribution of formate dehydrogenase and pointed out some unique features. Conversion of formate to CO₂ was detected, but labeled formate predominantly labeled the methyl group of acetate. Labeled CO₂ readily exchanged with the carboxyl group of pyruvate but not with formate, and both labeled CO₂ and pyruvate predominantly labeled the carboxyl group of acetate. No CO₂ was formed from O demethylation of vanillate, and the acetate produced was position labeled in the methyl group. The fermentation pattern and specific activities of products indicated a complete synthesis of acetate from pyruvate and the methoxyl group of vanillate.     

Tetrapyrrole biosynthesis in Anacystis nidulans; incorporation of [1-13C]-, [2-13C]-, [1,2-13C]- and [2-13C, 2-2H3]acetate

Labelling experiments with [2-¹³C]- and [1,2-¹³C]acetate showed that both photopigments of Anacystis nidulans, chlorophyll a and phycocyanobilin, share a common biosynthetic pathway from glutamate. The fate of deuterium during these biosynthetic events was studied using [2-¹³C, 2-²H₃]acetate as a precursor and determining the labelling pattern by ¹³C NMR spectroscopy with simultaneous [¹H, ²H]-broadband decoupling. The loss of ²H (ca 20%) from the precursor occurred at an early stage during the tricarboxylic acid cycle. After formation of glutamate there was no further loss of ²H in the assembly of the cyclic tetrapyrrole intermediates or during decarboxylation and modification of the side-chains. Thus the labelling data support a divergence in the pathway to cyclic and linear tetrapyrroles after protoporphyrin IX.     

Discontinuous replication of colicin E1 plasmid DNA in a cell extract containing thermolabile DNA ligase.

A cell extract prepared from the lig-ts7 mutant of Escherichia coli is able to carry out a complete round of DNA replication of colicin E1 plasmid at 25 °C. However, the apparent rate of elongation of the progeny strands at this temperature is much smaller than in an extract from the thermoresistant revertant cells. Chain elongation in the lig-ts extract is depressed by raising the incubation temperature from 25 °C to 32 °C, whereas that in the lig⁺ revertant extract is not. The rate of closure of the progeny strands of newly formed open circular molecules is also reduced in the lig-ts extract, even at 25 °C.The DNA pulse-labelled with the lig-ts extract for 30 seconds at 32 °C contains a large amount of short DNA fragments of approximately 7 S, in addition to DNA chains of various sizes between 7 S and 17 S (unit length). Most of these replicating molecules are converted to completely replicated closed circular molecules upon chasing with a lig⁺ extract. DNA-DNA hybridization experiments show that molecules replicated to various extents contain 7 S DNA fragments of both strands, but more of the L-strand component, whose 5′-to-3′ direction corresponds to the overall direction of unidirectional replication. The longer DNA chains are enriched in the H-strand component.The cell extracts used for the plasmid DNA replication have an activity which converts alkali-labile closed circular plasmid DNA containing apurinic sites to alkali-stable closed circular molecules. Addition of nicotinamide mononucleotide leads to conversion of the alkali-labile DNA to open circular molecules. In the replication system with the cell extract, however, the compound does not interfere with elongation of progeny strands. Chain elongation in the lig-ts extract at 25 °C is not significantly affected by nicotinamide mononucleotide. Thus, the 7 S DNA fragments formed with the lig-ts extract are unlikely to be generated as a result of incomplete repair of misincorporated nucleotides. We conclude that both strands of colicin E1 plasmid DNA replicate discontinuously.