A Total Synthesis of (±)-Gibberellin A12 via Methyl (±)-7,16-dioxo-17-norkauran-19-oate

¹⁴C-labelled methionine, xanthosine, and 7-methylxan-thosine were given to excised tea shoots. The methyl group of methionine was incorporated into 7-methylxanthosine (ca. 10%) in the earlier period of incubation after the uptake. About 50% of the radioactivity of xanthosine was rapidly incorporated into caffeine via 7-methylxanthosine, 7-methylxanthine, and theobromine within 24 hr. 7-Methylxanthosine was also converted into caffeine at a high rate. The results suggest that the pathway for caffeine biosynthesis is as follows: xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine.     

The biosynthesis of caffeine in the coffee plant

Leaf disks of Coffea arabica were infiltrated simultaneously with L-methionine-(methyl-¹⁴C) and with various possible precursors of caffeine biosynthesis. The results permit the identification of theobromine, 7-methylxanthine and 7-methylxanthosine as precursors of caffeine. 7-methylguanosine seems not to be an intermediate in caffeine formation.     

Metabolism of purine bases, nucleosides and alkaloids in theobromine-forming Theobroma cacao leaves

We examined the purine alkaloid content and purine metabolism in cacao (Theobroma cacao L.) plant leaves at various ages: young small leaves (stage I), developing intermediate size leaves (stage II), fully developed leaves (stage III) from flush shoots, and aged leaves (stage IV) from 1-year-old shoots. The major purine alkaloid in stage I leaves was theobromine (4.5 μmol g^–1 fresh weight), followed by caffeine (0.75 μmol g^–1 fresh weight). More than 75% of purine alkaloids disappeared with subsequent leaf development (stages II–IV). In stage I leaves, ¹⁴C-labelled adenine, adenosine, guanine, guanosine, hypoxanthine and inosine were converted to salvage products (nucleotides and nucleic acids), to degradation products (ureides and CO₂) and to purine alkaloids (3- and 7-methylxanthine, 7-methylxanthosine and theobromine). In contrast, ¹⁴C-labelled xanthine and xanthosine were not used for nucleotide synthesis. They were completely degraded, but nearly 20% of [8-¹⁴C]Xanthosine was converted in stage I leaves to purine alkaloids. These observations are consistent with the following biosynthetic pathways for theobromine: (a) AMP → IMP → 5′-xanthosine monophosphate → xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine; (b) GMP → guanosine → xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine; (c) xanthine → 3-methylxanthine → theobromine. Although no caffeine biosynthesis from ¹⁴C-labelled purine bases and nucleosides was observed during 18 h incubations, exogenously supplied [8-¹⁴C]Theobromine was converted to caffeine in young leaves. Conversion of theobromine to caffeine may, therefore, be slow in cacao leaves. No purine alkaloid synthesis was observed in the subsequent growth stages (stages II–IV). Significant degradation of purine alkaloids was found in leaves of stages II and III, in which [8-¹⁴C]Theobromine was degraded to CO₂ via 3-methylxanthine, xanthine and allantoic acid. [8-¹⁴C]Caffeine was catabolised to CO₂ via theophylline (1,3-dimethylxanthine) or theobromine.     

Degradation of caffeine and related methylxanthines bySerratia marcescens isolated from soil under coffee cultivation

A strain of Serratia marcescens showing the ability to degrade caffeine and other methylxanthines was isolated from soil under coffee cultivation. Growth was observed only with xanthines methylated at the 7 position (caffeine, 1,3,7-dimethylxanthine; paraxanthine, 1,7-dimethylxanthine; theobromine, 3,7-dimethylxanthine and 7-methylxanthine). Paraxanthine and theobromine were released in liquid medium when caffeine was used as the sole source of carbon and nitrogen. When paraxanthine or theobromine were used, 3-methylxanthine, 7-methylxanthine, and xanthine were detected in the liquid medium. Serratia marcescens did not grow with theophylline (1,3-dimethylxanthine), 1-methylxanthine, and 3-methylxanthine, and poor growth was observed with xanthine. Methyluric acid formation from methylxanthines was tested in cell-free extracts by measuring dehydrogenase reduction of tetrazolium salt in native-polyacrylamide gel electrophoresis gel. Activity was observed for all methylxanthines, even those with which no bacterial growth was observed. Our results suggest that in this strain of S. marcescens caffeine is degraded to theobromine (3,7-dimethylxanthine) and/or paraxanthine (1,7-dimethylxanthine), and subsequently to 7-methylxanthine and xanthine. Methyluric acid formation could not be confirmed. Correspondence to: Paulo Mazzafera.     

Fine control of caffeine biosynthesis in tissue cultures of Camellia sinensis

To determine whether caffeine biosynthesis is controlled by the availability of purine precursors and/or methyl-donors, we examined the effect of some purine compounds on purine alkaloid accumulation, using tea callus cultures. No stimulation of caffeine biosynthesis was observed when the calli were cultured with 0.5 mM adenosine, guanosine or hypoxanthine for 3 weeks. However, 0.5 mM paraxanthine doubled the caffeine level relative to controls. Adenosine stimulated the growth of callus and reduced the caffeine concentration 3 months after inoculation. These results indicate that methylation of xanthosine by 7-methylxanthosine synthase is the most plausible rate-limiting step of caffeine biosynthesis; the supply of non-methylated purine precursors or availability of S-adenosyl-l-methionine are not the principal controlling factors of caffeine biosynthesis. Adenosine salvage to adenine nucleotide synthesis may contribute to the growth of tea calli, but not to caffeine biosynthesis.     

Biosynthesis of caffeine by tea-leaf extracts. Enzymic formation of theobromine from 7-methylxanthine and of caffeine from theobromine.

1. Extracts prepared from tea leaves with Polyclar AT (insoluble polyvinylpyrrolidine) contained two methyltransferase activities catalysing the transfer of methyl groups from S-adenosylmethionine to 7-methylxanthine, producing theobromine, and to theobromine, producing caffeine. 2. The methyltransferases exhibited the same pH optimum (8.4) and a similar pattern of effects by metal ions, thiol inhibitors and metal-chelating reagents, both for theobromine and caffeine synthesis. Mg2+, Mn2+ and Ca2+ slightly stimulated enzyme activity but they were not essential. Paraxanthine was shown to be most active among methylxanthines, as the methyl acceptor. However, the formation of paraxanthine from 1-methylxanthine was very low and that from 7-methylxanthine was nil, suggesting that the synthesis of caffeine from paraxanthine is of little importance in intact plants. Xanthine, xanthosine, XMP and hypoxanthine were all inactive as methyl acceptors, whereas [2(-14)C]xanthine and [8(-14)C]hypoxanthine were catabolized to allantoin and urea by tea-leaf extracts. The apparent Km values are as follows: 7-methylxanthine, 1.0 times 10(-14)M; theobromine, 1.0 times 10(-3)M; paraxanthine, 0.2 times 10(-3)M; S-adenosylmethionine, 0.25 times 10(-4)M (with each of the three substrates). 3. The results suggest that the pathway for caffeine biosynthesis is as follows: 7-methylxanthine leads to theobromine leads to caffeine. In contrast, it is suggested that theophylline is synthesized from 1-methylxanthine. The methyl groups of the purine ring of caffeine are all derived directly from the methyl group of S-adenosylmethionine. Little is known about the pathways leading to the formation of 7-methylxanthine. 4. A good correlation between caffeine synthesis and shoot formation or growth of tea seedlings was shown, suggesting that the methylating systems in caffeine synthesis are closely associated with purine nucleotide and nucleic acid metabolism in tea plants.     

Caffeine biosynthesis in young leaves of Camellia sinensis: In vitro studies on N-methyltransferase activity involved in the conversion of xanthosine to caffeine

The aim of this study was to investigate the S -adenosylmethionine dependent N -methyltransferase(s) (NMT) associated with the three methylation steps in the caffeine biosynthesis pathway in tea ( Camellia sinensis L.). NMT activity in cell-free preparations from young leaves was purified by anion-exchange and gel-filtration column chromatography. In both systems, a single zone of NMT activity, with broad substrate specificity was detected. The N-3 position of dimethylxanthine and monomethylxanthines was methylated more readily than N-1 while comparatively little substitution occurred at the N-7 locus. When xanthosine was used as a substrate only the N-7 position was methylated. These results indicate that a single NMT may participate in the conversion of xanthosine to caffeine. The apparent M_r of the NMT, estimated by gel filtration chromatography, was 61 000. The substrate specificity of the NMT is compatible with the operation of a xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine pathway as the main biosynthetic route to caffeine in young tea leaves. The data also indicate that the conversion of 7-methylxanthine → paraxanthine → caffeine may function as one of a number of minor pathways that also contribute to the production of caffeine.     

Caffeine biosynthesis in young leaves of Camellia sinensis: In vitro studies on N-methyltransferase activity involved in the conversion of xanthosine to caffeine

The aim of this study was to investigate the S -adenosylmethionine dependent N -methyltransferase(s) (NMT) associated with the three methylation steps in the caffeine biosynthesis pathway in tea ( Camellia sinensis L.). NMT activity in cell-free preparations from young leaves was purified by anion-exchange and gel-filtration column chromatography. In both systems, a single zone of NMT activity, with broad substrate specificity was detected. The N-3 position of dimethylxanthine and monomethylxanthines was methylated more readily than N-1 while comparatively little substitution occurred at the N-7 locus. When xanthosine was used as a substrate only the N-7 position was methylated. These results indicate that a single NMT may participate in the conversion of xanthosine to caffeine. The apparent M_r of the NMT, estimated by gel filtration chromatography, was 61 000. The substrate specificity of the NMT is compatible with the operation of a xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine pathway as the main biosynthetic route to caffeine in young tea leaves. The data also indicate that the conversion of 7-methylxanthine → paraxanthine → caffeine may function as one of a number of minor pathways that also contribute to the production of caffeine.     

Catabolism of caffeine and related purine alkaloids in leaves ofCoffea arabica L.

In a study of purine alkaloid catabolism pathways in coffee,¹⁴C-labelled theobromine, caffeine, theophylline and xanthine were incubated with leaves ofCoffea arabica. Incorporation of label into¹⁴CO₂ was determined and methanol-soluble metabolites were analysed by high-performance liquid chromatography-radiocounting. The data obtained demonstrate catabolism of caffeine theophylline 3-methylxanthine xanthine. Xanthine is degraded further by the conventional purine catabolism pathway to CO₂ and NH₃ via uric acid, allantoin and allantoic acid. The conversion of caffeine to theophylline is the rate-limiting step in purine alkaloid catabolism and provides a ready explanation for the high concentration of endogenous caffeine found inC. arabica leaves. Although theobromine is converted primarily to caffeine, a small portion of the theobromine pool appears to be degraded to xanthine by a caffeine-independent pathway. In addition to being broken down to CO₂, via the purine catabolism pathway, xanthine is metabolised to 7-methylxanthine. Metabolism of [2-¹⁴C]xanthine byC. arabica leaves in the presence of 5 mM allopurinol results in very large increases in incorporation of radioactivity into 7-methylxanthine as degradation of the substrate via the purine catabolism pathway is blocked. The identity of 7-methylxanthine in these studies was confirmed by gas chromatography-mass spectrometry analysis.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting This work was supported by the British Council which provided H.A. with Japan-UK travel grants. F.M.G. was supported by a Biotechnology and Biological Sciences Research Council grant to A.C.     

Degradation Kinetics of Caffeine and Related Methylxanthines by Induced Cells of <Emphasis Type="Italic">Pseudomonas</Emphasis> sp.

In this study, the kinetics of degradation of caffeine and related methylxanthines by induced cells of Pseudomonas sp. was performed. The kinetics data showed that degradation of caffeine, theobromine, and 7-methylxanthine followed Michealis–Menten kinetics. The values of K _m are low for caffeine and 7-methylxanthine and high for theobromine. Degradation of caffeine and theobromine was enhanced in the presence of NADH and NADPH, whereas the degradation of 7-methylxanthine was unaffected. Among the various metal ions tested, Fe²⁺ was found to enhance the rate of degradation for all three substrates, whereas Zn²⁺ and Cu²⁺ inhibited the degradation of caffeine and theobromine but not 7-methylxanthine. The differences in kinetic parameters and cofactor requirement suggest the possibility of the involvement of more than one N-demethylases in the caffeine catabolic pathway in Pseudomonas sp. The induced cells can serve as effective biocatalysts for the development of biodecaffeination techniques.     

Purification and characterization of caffeine synthase from tea leaves.

Caffeine synthase (CS), the S-adenosylmethionine-dependent N-methyltransferase involved in the last two steps of caffeine biosynthesis, was extracted from young tea (Camellia sinensis) leaves; the CS was purified 520-fold to apparent homogeneity and a final specific activity of 5.7 nkat mg-1 protein by ammonium sulfate fractionation and hydroxyapatite, anion-exchange, adenosine-agarose, and gel-filtration chromatography. The native enzyme was monomeric with an apparent molecular mass of 61 kD as estimated by gel-filtration chromatography and 41 kD as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme displayed a sharp pH optimum of 8.5. The final preparation exhibited 3- and 1-N-methyltransferase activity with a broad substrate specificity, showing high activity toward paraxanthine, 7-methylxanthine, and theobromine and low activity with 3-methylxanthine and 1-methylxanthine. However, the enzyme had no 7-N-methyltransferase activity toward xanthosine and xanthosine 5'-monophosphate. The Km values of CS for paraxanthine, theobromine, 7-methylxanthine, and S-adenosylmethionine were 24, 186, 344, and 21 microM, respectively. The possible role and regulation of CS in purine alkaloid biosynthesis in tea leaves are discussed. The 20-amino acid N-terminal sequence for CS showed little homology with other methyltransferases.     

Engineering a microbial platform for de novo biosynthesis of diverse methylxanthines

Engineered microbial biosynthesis of plant natural products can support manufacturing of complex bioactive molecules and enable discovery of non-naturally occurring derivatives. Purine alkaloids, including caffeine (coffee), theophylline (antiasthma drug), theobromine (chocolate), and other methylxanthines, play a significant role in pharmacology and food chemistry. Here, we engineered the eukaryotic microbial host Saccharomyces cerevisiae for the de novo biosynthesis of methylxanthines. We constructed a xanthine-to-xanthosine conversion pathway in native yeast central metabolism to increase endogenous purine flux for the production of 7-methylxanthine, a key intermediate in caffeine biosynthesis. Yeast strains were further engineered to produce caffeine through expression of several enzymes from the coffee plant. By expressing combinations of different N-methyltransferases, we were able to demonstrate re-direction of flux to an alternate pathway and develop strains that support the production of diverse methylxanthines. We achieved production of 270 μg/L, 61 μg/L, and 3700 μg/L of caffeine, theophylline, and 3-methylxanthine, respectively, in 0.3-L bench-scale batch fermentations. The constructed strains provide an early platform for de novo production of methylxanthines and with further development will advance the discovery and synthesis of xanthine derivatives.     

Biosynthesis of Caffeine in Leaves of Coffee

The levels of endogenous caffeine and theobromine were much higher in buds and young leaves of Coffea arabica L. cv Kent than in fully developed leaves. Biosynthesis of caffeine from 14C-labeled adenine, guanine, xanthosine, and theobromine was observed, whereas other studies (H. Ashihara, A.M. Monteiro, T. Moritz, F.M. Gillies, A. Crozier [1996] Planta 198: 334-339) have indicated that there is no detectable incorporation of label into caffeine when theophylline and xanthine are used as substrates for in vivo feeds with leaves of C. arabica. The capacity for caffeine biosynthesis, especially from guanine and xanthosine, was reduced markedly in both fully developed mature and aged leaves. Data obtained in pulse-chase experiments with young leaves indicate the operation of an AMP -> IMP -> xanthosine 5[prime]-monophosphate (or GMP -> guanosine) -> xanthosine -> 7-methylxanthosine -> 7-methylxanthine -> theobromine -> caffeine pathway. The data obtained provide strong evidence against proposals by G.M. Nazario and C.J. Lovatt ([1993] Plant Physiol 103: 1203-1210) concerning the independence of caffeine and theobromine biosynthesis pathways and the role of xanthine as a key intermediate in caffeine biosynthesis.     

Novel, highly specific N-demethylases enable bacteria to live on caffeine and related purine alkaloids

The molecular basis for the ability of bacteria to live on caffeine as a sole carbon and nitrogen source is unknown. Pseudomonas putida CBB5, which grows on several purine alkaloids, metabolizes caffeine and related methylxanthines via sequential N-demethylation to xanthine. Metabolism of caffeine by CBB5 was previously attributed to one broad-specificity methylxanthine N-demethylase composed of two subunits, NdmA and NdmB. Here, we report that NdmA and NdmB are actually two independent Rieske nonheme iron monooxygenases with N(1)- and N(3)-specific N-demethylation activity, respectively. Activity for both enzymes is dependent on electron transfer from NADH via a redox-center-dense Rieske reductase, NdmD. NdmD itself is a novel protein with one Rieske [2Fe-2S] cluster, one plant-type [2Fe-2S] cluster, and one flavin mononucleotide (FMN) per enzyme. All ndm genes are located in a 13.2-kb genomic DNA fragment which also contained a formaldehyde dehydrogenase. ndmA, ndmB, and ndmD were cloned as His(6) fusion genes, expressed in Escherichia coli, and purified using a Ni-NTA column. NdmA-His(6) plus His(6)-NdmD catalyzed N(1)-demethylation of caffeine, theophylline, paraxanthine, and 1-methylxanthine to theobromine, 3-methylxanthine, 7-methylxanthine, and xanthine, respectively. NdmB-His(6) plus His(6)-NdmD catalyzed N(3)-demethylation of theobromine, 3-methylxanthine, caffeine, and theophylline to 7-methylxanthine, xanthine, paraxanthine, and 1-methylxanthine, respectively. One formaldehyde was produced from each methyl group removed. Activity of an N(7)-specific N-demethylase, NdmC, has been confirmed biochemically. This is the first report of bacterial N-demethylase genes that enable bacteria to live on caffeine. These genes represent a new class of Rieske oxygenases and have the potential to produce biofuels, animal feed, and pharmaceuticals from coffee and tea waste.     

Two Distinct Pathways for Metabolism of Theophylline and Caffeine Are Coexpressed in Pseudomonas putida CBB5

Pseudomonas putida CBB5 was isolated from soil by enrichment on caffeine. This strain used not only caffeine, theobromine, paraxanthine, and 7-methylxanthine as sole carbon and nitrogen sources but also theophylline and 3-methylxanthine. Analyses of metabolites in spent media and resting cell suspensions confirmed that CBB5 initially N demethylated theophylline via a hitherto unreported pathway to 1- and 3-methylxanthines. NAD(P)H-dependent conversion of theophylline to 1- and 3-methylxanthines was also detected in the crude cell extracts of theophylline-grown CBB5. 1-Methylxanthine and 3-methylxanthine were subsequently N demethylated to xanthine. CBB5 also oxidized theophylline and 1- and 3-methylxanthines to 1,3-dimethyluric acid and 1- and 3-methyluric acids, respectively. However, these methyluric acids were not metabolized further. A broad-substrate-range xanthine-oxidizing enzyme was responsible for the formation of these methyluric acids. In contrast, CBB5 metabolized caffeine to theobromine (major metabolite) and paraxanthine (minor metabolite). These dimethylxanthines were further N demethylated to xanthine via 7-methylxanthine. Theobromine-, paraxanthine-, and 7-methylxanthine-grown cells also metabolized all of the methylxanthines mentioned above via the same pathway. Thus, the theophylline and caffeine N-demethylation pathways converged at xanthine via different methylxanthine intermediates. Xanthine was eventually oxidized to uric acid. Enzymes involved in theophylline and caffeine degradation were coexpressed when CBB5 was grown on theophylline or on caffeine or its metabolites. However, 3-methylxanthine-grown CBB5 cells did not metabolize caffeine, whereas theophylline was metabolized at much reduced levels to only methyluric acids. To our knowledge, this is the first report of theophylline N demethylation and coexpression of distinct pathways for caffeine and theophylline degradation in bacteria.Caffeine (1,3,7-trimethylxanthine) and related methylxanthines are widely distributed in many plant species. Caffeine is also a major human dietary ingredient that can be found in common beverages and food products, such as coffee, tea, and chocolates. In pharmaceuticals, caffeine is used generally as a cardiac, neurological, and respiratory stimulant, as well as a diuretic (3). Hence, caffeine and related methylxanthines enter soil and water easily through decomposed plant materials and other means, such as effluents from coffee- and tea-processing facilities. Therefore, it is not surprising that microorganisms capable of degrading caffeine have been isolated from various natural environments, with or without enrichment procedures (3, 10). Bacteria use oxidative and N-demethylating pathways for catabolism of caffeine. Oxidation of caffeine by a Rhodococcus sp.-Klebsiella sp. mixed-culture consortium at the C-8 position to form 1,3,7-trimethyluric acid (TMU) has been reported (8). An 85-kDa, flavin-containing caffeine oxidase was purified from this consortium (9). Also, Mohapatra et al. (12) purified a 65-kDa caffeine oxidase from Alcaligenes sp. strain CF8. Cells of a caffeine-degrading Pseudomonas putida strain (ATCC 700097) isolated from domestic wastewater (13) showed a fourfold increase in a cytochrome P450 absorption spectrum signal compared to cells grown on glucose. Recently, we reported a novel non-NAD(P)⁺-dependent heterotrimeric caffeine dehydrogenase from Pseudomonas sp. strain CBB1 (20). This enzyme oxidized caffeine to TMU stoichiometrically and hydrolytically, without producing hydrogen peroxide. Further metabolism of TMU has not been elucidated.Several caffeine-degrading bacteria metabolize caffeine via the N-demethylating pathway and produce theobromine (3,7-dimethylxanthine) or paraxanthine (1,7-dimethylxanthine) as the initial product. Theophylline (1,3-dimethylxanthine) has not been reported to be a metabolite in bacterial degradation of caffeine. Subsequent N demethylation of theobromine or paraxanthine to xanthine is via 7-methyxanthine. Xanthine is further oxidized to uric acid by xanthine dehydrogenase/oxidase (3, 10). Although the identities of metabolites and the sequence of metabolite formation for caffeine N demethylation are well established, there is very little information on the number and nature of N-demethylases involved in this pathway.The lack of adequate information on the metabolism and enzymology of theophylline, caffeine, and related methylxanthines prompted us to investigate the degradation of these compounds in detail. We isolated a unique caffeine-degrading bacterium, P. putida CBB5, from soil via enrichment with caffeine as the sole source of carbon and nitrogen. Here we describe a detailed study of the metabolism of theophylline, caffeine, and related di- and monomethylxanthines by CBB5. Our results indicate that CBB5 initially N demethylated caffeine to produce theobromine (major product) and paraxanthine (minor product) before the pathways converged to 7-methylxanthine and xanthine. Surprisingly, CBB5 was also capable of utilizing theophylline as a sole carbon and nitrogen source. CBB5 N demethylated theophylline to 1-methylxanthine and 3-methylxanthine, which were further N demethylated to xanthine. Theophylline N-demethylase activity was detected in cell extracts prepared from theophylline-grown CBB5 cells. 1-Methylxanthine and 3-methylxanthine were detected as products of this NAD(P)H-dependent reaction. To our knowledge, this is the first report of a theophylline degradation pathway in bacteria and coexpression of distinct caffeine and theophylline degradation pathways.     

Chronic Effects of Xanthines on Levels of Central Receptors in Mice

1.Chronic ingestion of caffeine causes a significant increase in levels of A₁-adenosine, nicotinic and muscarinic receptors, serotonergic receptors, GABA_A receptors and L-type calcium channels in cerebral cortical membranes from mice NIH Swiss strain mice.2.Chronic theophylline and paraxanthine had effects similar to those of caffeine except that levels of L-type channels were unchanged. Chronic theobromine, a weak adenosine antagonist, and 1-isobutyl-3-methylxanthine (IBMX), a potent adenosine antagonist and phosphodiesterase inhibitor, caused only an increase in levels of A₁-adenosine receptors. A combination of chronic caffeine and IBMX had the same effects on receptors as caffeine alone. Chronic 3,7-dimethyl-1-propargylxanthine (DMPX), a somewhat selective A_2A-antagonist, caused only an increase in levels of A₁-adenosine receptors. Pentoxyfylline, an adenosine-uptake inhibitor inactive at adenosine receptors, had no effect on receptor levels or calcium channels.3.A comparison of plasma and brain levels of xanthines indicated that caffeine penetrated more readily and attained somewhat higher brain levels than theophylline or theobromine. Penetration and levels were even lower for IBMX, paraxanthine, DMPX, and pentoxyfylline.4.The results suggest that effective blockade of both A₁ and A_2A-adenosine receptors is necessary for the full spectrum of biochemical changes elicited by chronic ingestion of xanthines, such as caffeine, theophylline, and paraxanthine.     

Metabolic Engineering of Saccharomyces cerevisiae for Caffeine and Theobromine Production

Caffeine (1, 3, 7-trimethylxanthine) and theobromine (3, 7-dimethylxanthine) are the major purine alkaloids in plants, e.g. tea (Camellia sinensis) and coffee (Coffea arabica). Caffeine is a major component of coffee and is used widely in food and beverage industries. Most of the enzymes involved in the caffeine biosynthetic pathway have been reported previously. Here, we demonstrated the biosynthesis of caffeine (0.38 mg/L) by co-expression of Coffea arabica xanthosine methyltransferase (CaXMT) and Camellia sinensis caffeine synthase (TCS) in Saccharomyces cerevisiae. Furthermore, we endeavored to develop this production platform for making other purine-based alkaloids. To increase the catalytic activity of TCS in an effort to increase theobromine production, we identified four amino acid residues based on structural analyses of 3D-model of TCS. Two TCS1 mutants (Val317Met and Phe217Trp) slightly increased in theobromine accumulation and simultaneously decreased in caffeine production. The application and further optimization of this biosynthetic platform are discussed.     

Caffeine biosynthesis in Camellia sinensis

The metabolism of adenine and guanine, relating to the biosynthesis of caffeine, in excised shoot tips of tea was studied with micromolar amounts of adenine-[8-¹⁴C] or guanine-[8-¹⁴C]. Among the presumed precursors of caffeine biosynthesis, adenine was the most effective, whereas guanine was the least effective. After administration of a ‘pulse’ of adenine-[8-¹⁴C], almost all of the adenine-[¹⁴C] supplied disappeared by 30 hr, and ¹⁴C-labelled caffeine and RNA purine nucleotide (AMP and GMP) synthesis increased throughout the experimental period, whereas the radioactivities of free purine nucleotides, 7-methylxanthine and theobromine increased during the first 10 hr incubation period, followed by a steady decrease. By contrast, more than 45% of the guanine-[8-¹⁴C] supplied remained unchanged even after a 120 hr period. The main products of guanine-[8-¹⁴C] metabolism in tea shoot tips were guanine nucleotides, theobromine, caffeine and the GMP of RNA. The results support the hypothesis that the purine nucleotides are synthesized from adenine and guanine via the pathway of purine salvage. Adenylate is readily converted into other purine nucleotides, whereas the conversion rate of guanylate into other purine nucleotides is very low.The results also support the view that 7-methylxanthine and theobromine are precursors of caffeine. For the origin of the purine ring in caffeine, purine nucleotides in the nucleotide pool rather than in nucleic acids are suggested.     

Biosynthesis and metabolism of purine alkaloids in leaves of cocoa tea (Camellia ptilophylla)

The biosynthesis and metabolism of purine alkaloids in leaves ofCamellia ptilophylla (cocoa tea), a new tea resource in China, have been investigated. The major purine alkaloid was theobromine, with theophylline also being present as a minor component. Caffeine was not accumulated in detectable quantities. Theobromine was synthesized from [8-¹⁴C] adenine and the rate of its biosynthesis in the segments from young and mature leaves from flush shoots was approximately 10 times higher than that from aged leaves from 1-year old shoots. Neither cellfree extracts nor segments fromC. ptilophylla leaves could convert theobromine to caffeine. A large quantity of [2-¹⁴C] xanthine taken up by the leaf segments was degraded to¹⁴CO₂ via the conventional purine catabolic pathway that includes allantoin as an intermediate. However, small amounts of [2-¹⁴C] xanthine were also converted to theobromine. Considerable amounts of [8-¹⁴C] caffeine exogenously supplied to the leaf segments ofC. ptilophylla was changed to theobromine. These results indicate that leaves ofC. ptilophylla exhibit unusual purine alkaloid metabolism as i) they have the capacity to synthesize theobromine from adenine nucleotides, but they lack adequate methyltransferase activity to convert of theobromine to caffeine in detectable quantities, ii) the leaves have a capacity to convert xanthine to theobromine, probably via 3-methylxanthine.