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Xiaohui Liu – One of the best experts on this subject based on the ideXlab platform.

  • 3 Methylxanthine production through biodegradation of theobromine by aspergillus sydowii pt 2
    BMC Microbiology, 2020
    Co-Authors: Bin-xing Zhou, Chengqin Zheng, Tao Xia, Xiaohui Liu
    Abstract:

    Methylxanthines, including caffeine, theobromine and theophylline, are natural and synthetic compounds in tea, which could be metabolized by certain kinds of bacteria and fungi. Previous studies confirmed that several microbial isolates from Pu-erh tea could degrade and convert caffeine and theophylline. We speculated that these candidate isolates also could degrade and convert theobromine through N-demethylation and oxidation. In this study, seven tea-derived fungal strains were inoculated into various theobromine agar medias and theobromine liquid mediums to assess their capacity in theobromine utilization. Related metabolites with theobromine degradation were detected by using HPLC in the liquid culture to investigate their potential application in the production of 3Methylxanthine. Based on theobromine utilization capacity, Aspergillus niger PT-1, Aspergillus sydowii PT-2, Aspergillus ustus PT-6 and Aspergillus tamarii PT-7 have demonstrated the potential for theobromine biodegradation. Particularly, A. sydowii PT-2 and A. tamarii PT-7 could degrade theobromine significantly (p < 0.05) in all given liquid mediums. 3,7-Dimethyluric acid, 3Methylxanthine, 7-Methylxanthine, 3-methyluric acid, xanthine, and uric acid were detected in A. sydowii PT-2 and A. tamarii PT-7 culture, respectively, which confirmed the existence of N-demethylation and oxidation in theobromine catabolism. 3Methylxanthine was common and main demethylated metabolite of theobromine in the liquid culture. 3Methylxanthine in A. sydowii PT-2 culture showed a linear relation with initial theobromine concentrations that 177.12 ± 14.06 mg/L 3Methylxanthine was accumulated in TLM-S with 300 mg/L theobromine. Additionally, pH at 5 and metal ion of Fe2+ promoted 3Methylxanthine production significantly (p < 0.05). This study is the first to confirm that A. sydowii PT-2 and A. tamarii PT-7 degrade theobromine through N-demethylation and oxidation, respectively. A. sydowii PT-2 showed the potential application in 3Methylxanthine production with theobromine as feedstock through the N-demethylation at N-7 position.

  • 3Methylxanthine production through biodegradation of theobromine by Aspergillus sydowii PT-2.
    BMC microbiology, 2020
    Co-Authors: Bin-xing Zhou, Chengqin Zheng, Tao Xia, Xiaohui Liu
    Abstract:

    Methylxanthines, including caffeine, theobromine and theophylline, are natural and synthetic compounds in tea, which could be metabolized by certain kinds of bacteria and fungi. Previous studies confirmed that several microbial isolates from Pu-erh tea could degrade and convert caffeine and theophylline. We speculated that these candidate isolates also could degrade and convert theobromine through N-demethylation and oxidation. In this study, seven tea-derived fungal strains were inoculated into various theobromine agar medias and theobromine liquid mediums to assess their capacity in theobromine utilization. Related metabolites with theobromine degradation were detected by using HPLC in the liquid culture to investigate their potential application in the production of 3Methylxanthine. Based on theobromine utilization capacity, Aspergillus niger PT-1, Aspergillus sydowii PT-2, Aspergillus ustus PT-6 and Aspergillus tamarii PT-7 have demonstrated the potential for theobromine biodegradation. Particularly, A. sydowii PT-2 and A. tamarii PT-7 could degrade theobromine significantly (p 

  • 3Methylxanthine production through biodegradation of theobromine by Aspergillus sydowii PT-2
    , 2020
    Co-Authors: Bin-xing Zhou, Chengqin Zheng, Tao Xia, Xiaohui Liu
    Abstract:

    Abstract Background: Methylxanthines, including caffeine, theobromine and theophylline, are natural and synthetic compounds in tea, which could be metabolized by certain kinds of bacteria and fungi. Previous studies confirmed that several microbial isolates from Pu-erh tea could degrade and convert caffeine and theophylline. We speculated that these candidate isolates also could degrade and convert theobromine through N-demethylation and oxidation. In this study, seven tea-derived fungal strains were inoculated into various theobromine agar medias and theobromine liquid mediums to assess their capacity in theobromine utilization. Related metabolites with theobromine degradation were detected by using HPLC in the liquid culture to investigate their potential application in the production of 3Methylxanthine. Results: Based on theobromine utilization capacity, Aspergillus niger PT-1, Aspergillus sydowii PT-2, Aspergillus ustus PT-6 and Aspergillus tamarii PT-7 have demonstrated the potential for theobromine biodegradation. Particularly, A. sydowii PT-2 and A. tamarii PT-7 could degrade theobromine significantly (p < 0.05) in all given liquid mediums. 3,7-Dimethyluric acid, 3Methylxanthine, 7-Methylxanthine, 3-methyluric acid, xanthine, and uric acid were detected in A. sydowii PT-2 and A. tamarii PT-7 culture, respectively, which confirmed the existence of N-demethylation and oxidation in theobromine catabolism. 3Methylxanthine was common and main demethylated metabolite of theobromine in the liquid culture. 3Methylxanthine in A. sydowii PT-2 culture showed a linear relation with initial theobromine concentrations that 177.12 ± 14.06 mg/L 3Methylxanthine was accumulated in TLM-S with 300 mg/L theobromine. Additionally, pH at 5 and metal ion of Fe2+ promoted 3Methylxanthine production significantly (p < 0.05). Conclusions: This study is the first to confirm that A. sydowii PT-2 and A. tamarii PT-7 degrade theobromine through N-demethylation and oxidation, respectively. A. sydowii PT-2 showed the potential application in 3Methylxanthine production with theobromine as feedstock through the N-demethylation at N-7 position.

Bin-xing Zhou – One of the best experts on this subject based on the ideXlab platform.

  • 3 Methylxanthine production through biodegradation of theobromine by aspergillus sydowii pt 2
    BMC Microbiology, 2020
    Co-Authors: Bin-xing Zhou, Chengqin Zheng, Tao Xia, Xiaohui Liu
    Abstract:

    Methylxanthines, including caffeine, theobromine and theophylline, are natural and synthetic compounds in tea, which could be metabolized by certain kinds of bacteria and fungi. Previous studies confirmed that several microbial isolates from Pu-erh tea could degrade and convert caffeine and theophylline. We speculated that these candidate isolates also could degrade and convert theobromine through N-demethylation and oxidation. In this study, seven tea-derived fungal strains were inoculated into various theobromine agar medias and theobromine liquid mediums to assess their capacity in theobromine utilization. Related metabolites with theobromine degradation were detected by using HPLC in the liquid culture to investigate their potential application in the production of 3Methylxanthine. Based on theobromine utilization capacity, Aspergillus niger PT-1, Aspergillus sydowii PT-2, Aspergillus ustus PT-6 and Aspergillus tamarii PT-7 have demonstrated the potential for theobromine biodegradation. Particularly, A. sydowii PT-2 and A. tamarii PT-7 could degrade theobromine significantly (p < 0.05) in all given liquid mediums. 3,7-Dimethyluric acid, 3Methylxanthine, 7-Methylxanthine, 3-methyluric acid, xanthine, and uric acid were detected in A. sydowii PT-2 and A. tamarii PT-7 culture, respectively, which confirmed the existence of N-demethylation and oxidation in theobromine catabolism. 3Methylxanthine was common and main demethylated metabolite of theobromine in the liquid culture. 3Methylxanthine in A. sydowii PT-2 culture showed a linear relation with initial theobromine concentrations that 177.12 ± 14.06 mg/L 3Methylxanthine was accumulated in TLM-S with 300 mg/L theobromine. Additionally, pH at 5 and metal ion of Fe2+ promoted 3Methylxanthine production significantly (p < 0.05). This study is the first to confirm that A. sydowii PT-2 and A. tamarii PT-7 degrade theobromine through N-demethylation and oxidation, respectively. A. sydowii PT-2 showed the potential application in 3Methylxanthine production with theobromine as feedstock through the N-demethylation at N-7 position.

  • 3Methylxanthine production through biodegradation of theobromine by Aspergillus sydowii PT-2.
    BMC microbiology, 2020
    Co-Authors: Bin-xing Zhou, Chengqin Zheng, Tao Xia, Xiaohui Liu
    Abstract:

    Methylxanthines, including caffeine, theobromine and theophylline, are natural and synthetic compounds in tea, which could be metabolized by certain kinds of bacteria and fungi. Previous studies confirmed that several microbial isolates from Pu-erh tea could degrade and convert caffeine and theophylline. We speculated that these candidate isolates also could degrade and convert theobromine through N-demethylation and oxidation. In this study, seven tea-derived fungal strains were inoculated into various theobromine agar medias and theobromine liquid mediums to assess their capacity in theobromine utilization. Related metabolites with theobromine degradation were detected by using HPLC in the liquid culture to investigate their potential application in the production of 3Methylxanthine. Based on theobromine utilization capacity, Aspergillus niger PT-1, Aspergillus sydowii PT-2, Aspergillus ustus PT-6 and Aspergillus tamarii PT-7 have demonstrated the potential for theobromine biodegradation. Particularly, A. sydowii PT-2 and A. tamarii PT-7 could degrade theobromine significantly (p 

  • 3Methylxanthine production through biodegradation of theobromine by Aspergillus sydowii PT-2
    , 2020
    Co-Authors: Bin-xing Zhou, Chengqin Zheng, Tao Xia, Xiaohui Liu
    Abstract:

    Abstract Background: Methylxanthines, including caffeine, theobromine and theophylline, are natural and synthetic compounds in tea, which could be metabolized by certain kinds of bacteria and fungi. Previous studies confirmed that several microbial isolates from Pu-erh tea could degrade and convert caffeine and theophylline. We speculated that these candidate isolates also could degrade and convert theobromine through N-demethylation and oxidation. In this study, seven tea-derived fungal strains were inoculated into various theobromine agar medias and theobromine liquid mediums to assess their capacity in theobromine utilization. Related metabolites with theobromine degradation were detected by using HPLC in the liquid culture to investigate their potential application in the production of 3Methylxanthine. Results: Based on theobromine utilization capacity, Aspergillus niger PT-1, Aspergillus sydowii PT-2, Aspergillus ustus PT-6 and Aspergillus tamarii PT-7 have demonstrated the potential for theobromine biodegradation. Particularly, A. sydowii PT-2 and A. tamarii PT-7 could degrade theobromine significantly (p < 0.05) in all given liquid mediums. 3,7-Dimethyluric acid, 3Methylxanthine, 7-Methylxanthine, 3-methyluric acid, xanthine, and uric acid were detected in A. sydowii PT-2 and A. tamarii PT-7 culture, respectively, which confirmed the existence of N-demethylation and oxidation in theobromine catabolism. 3Methylxanthine was common and main demethylated metabolite of theobromine in the liquid culture. 3Methylxanthine in A. sydowii PT-2 culture showed a linear relation with initial theobromine concentrations that 177.12 ± 14.06 mg/L 3Methylxanthine was accumulated in TLM-S with 300 mg/L theobromine. Additionally, pH at 5 and metal ion of Fe2+ promoted 3Methylxanthine production significantly (p < 0.05). Conclusions: This study is the first to confirm that A. sydowii PT-2 and A. tamarii PT-7 degrade theobromine through N-demethylation and oxidation, respectively. A. sydowii PT-2 showed the potential application in 3Methylxanthine production with theobromine as feedstock through the N-demethylation at N-7 position.

Tao Xia – One of the best experts on this subject based on the ideXlab platform.

  • 3 Methylxanthine production through biodegradation of theobromine by aspergillus sydowii pt 2
    BMC Microbiology, 2020
    Co-Authors: Bin-xing Zhou, Chengqin Zheng, Tao Xia, Xiaohui Liu
    Abstract:

    Methylxanthines, including caffeine, theobromine and theophylline, are natural and synthetic compounds in tea, which could be metabolized by certain kinds of bacteria and fungi. Previous studies confirmed that several microbial isolates from Pu-erh tea could degrade and convert caffeine and theophylline. We speculated that these candidate isolates also could degrade and convert theobromine through N-demethylation and oxidation. In this study, seven tea-derived fungal strains were inoculated into various theobromine agar medias and theobromine liquid mediums to assess their capacity in theobromine utilization. Related metabolites with theobromine degradation were detected by using HPLC in the liquid culture to investigate their potential application in the production of 3Methylxanthine. Based on theobromine utilization capacity, Aspergillus niger PT-1, Aspergillus sydowii PT-2, Aspergillus ustus PT-6 and Aspergillus tamarii PT-7 have demonstrated the potential for theobromine biodegradation. Particularly, A. sydowii PT-2 and A. tamarii PT-7 could degrade theobromine significantly (p < 0.05) in all given liquid mediums. 3,7-Dimethyluric acid, 3Methylxanthine, 7-Methylxanthine, 3-methyluric acid, xanthine, and uric acid were detected in A. sydowii PT-2 and A. tamarii PT-7 culture, respectively, which confirmed the existence of N-demethylation and oxidation in theobromine catabolism. 3Methylxanthine was common and main demethylated metabolite of theobromine in the liquid culture. 3Methylxanthine in A. sydowii PT-2 culture showed a linear relation with initial theobromine concentrations that 177.12 ± 14.06 mg/L 3Methylxanthine was accumulated in TLM-S with 300 mg/L theobromine. Additionally, pH at 5 and metal ion of Fe2+ promoted 3Methylxanthine production significantly (p < 0.05). This study is the first to confirm that A. sydowii PT-2 and A. tamarii PT-7 degrade theobromine through N-demethylation and oxidation, respectively. A. sydowii PT-2 showed the potential application in 3Methylxanthine production with theobromine as feedstock through the N-demethylation at N-7 position.

  • 3Methylxanthine production through biodegradation of theobromine by Aspergillus sydowii PT-2.
    BMC microbiology, 2020
    Co-Authors: Bin-xing Zhou, Chengqin Zheng, Tao Xia, Xiaohui Liu
    Abstract:

    Methylxanthines, including caffeine, theobromine and theophylline, are natural and synthetic compounds in tea, which could be metabolized by certain kinds of bacteria and fungi. Previous studies confirmed that several microbial isolates from Pu-erh tea could degrade and convert caffeine and theophylline. We speculated that these candidate isolates also could degrade and convert theobromine through N-demethylation and oxidation. In this study, seven tea-derived fungal strains were inoculated into various theobromine agar medias and theobromine liquid mediums to assess their capacity in theobromine utilization. Related metabolites with theobromine degradation were detected by using HPLC in the liquid culture to investigate their potential application in the production of 3Methylxanthine. Based on theobromine utilization capacity, Aspergillus niger PT-1, Aspergillus sydowii PT-2, Aspergillus ustus PT-6 and Aspergillus tamarii PT-7 have demonstrated the potential for theobromine biodegradation. Particularly, A. sydowii PT-2 and A. tamarii PT-7 could degrade theobromine significantly (p 

  • 3Methylxanthine production through biodegradation of theobromine by Aspergillus sydowii PT-2
    , 2020
    Co-Authors: Bin-xing Zhou, Chengqin Zheng, Tao Xia, Xiaohui Liu
    Abstract:

    Abstract Background: Methylxanthines, including caffeine, theobromine and theophylline, are natural and synthetic compounds in tea, which could be metabolized by certain kinds of bacteria and fungi. Previous studies confirmed that several microbial isolates from Pu-erh tea could degrade and convert caffeine and theophylline. We speculated that these candidate isolates also could degrade and convert theobromine through N-demethylation and oxidation. In this study, seven tea-derived fungal strains were inoculated into various theobromine agar medias and theobromine liquid mediums to assess their capacity in theobromine utilization. Related metabolites with theobromine degradation were detected by using HPLC in the liquid culture to investigate their potential application in the production of 3Methylxanthine. Results: Based on theobromine utilization capacity, Aspergillus niger PT-1, Aspergillus sydowii PT-2, Aspergillus ustus PT-6 and Aspergillus tamarii PT-7 have demonstrated the potential for theobromine biodegradation. Particularly, A. sydowii PT-2 and A. tamarii PT-7 could degrade theobromine significantly (p < 0.05) in all given liquid mediums. 3,7-Dimethyluric acid, 3Methylxanthine, 7-Methylxanthine, 3-methyluric acid, xanthine, and uric acid were detected in A. sydowii PT-2 and A. tamarii PT-7 culture, respectively, which confirmed the existence of N-demethylation and oxidation in theobromine catabolism. 3Methylxanthine was common and main demethylated metabolite of theobromine in the liquid culture. 3Methylxanthine in A. sydowii PT-2 culture showed a linear relation with initial theobromine concentrations that 177.12 ± 14.06 mg/L 3Methylxanthine was accumulated in TLM-S with 300 mg/L theobromine. Additionally, pH at 5 and metal ion of Fe2+ promoted 3Methylxanthine production significantly (p < 0.05). Conclusions: This study is the first to confirm that A. sydowii PT-2 and A. tamarii PT-7 degrade theobromine through N-demethylation and oxidation, respectively. A. sydowii PT-2 showed the potential application in 3Methylxanthine production with theobromine as feedstock through the N-demethylation at N-7 position.

Mani Subramanian – One of the best experts on this subject based on the ideXlab platform.

  • Direct conversion of theophylline to 3Methylxanthine by metabolically engineered E. coli
    Microbial Cell Factories, 2015
    Co-Authors: Khalid H. R. Algharrawi, Ryan M. Summers, Sridhar Gopishetty, Mani Subramanian
    Abstract:

    Background Methylxanthines are natural and synthetic compounds found in many foods, drinks, pharmaceuticals, and cosmetics. Aside from caffeine, production of many Methylxanthines is currently performed by chemical synthesis. This process utilizes many chemicals, multiple reactions, and different reaction conditions, making it complicated, environmentally dissatisfactory, and expensive, especially for monoMethylxanthines and paraxanthine. A microbial platform could provide an economical, environmentally friendly approach to produce these chemicals in large quantities. The recently discovered genes in our laboratory from Pseudomonas putida , ndmA, ndmB , and ndmD , provide an excellent starting point for precisely engineering Escherichia coli with various gene combinations to produce specific high-value paraxanthine and 1-, 3-, and 7-Methylxanthines from any of the economical feedstocks including caffeine, theobromine or theophylline. Here, we show the first example of direct conversion of theophylline to 3Methylxanthine by a metabolically engineered strain of E. coli . Results Here we report the construction of E. coli strains with ndmA and ndmD , capable of producing 3Methylxanthine from exogenously fed theophylline. The strains were engineered with various dosages of the ndmA and ndmD genes, screened, and the best strain was selected for large-scale conversion of theophylline to 3Methylxanthine. Strain pDdA grown in super broth was the most efficient strain; 15 mg/mL cells produced 135 mg/L (0.81 mM) 3Methylxanthine from 1 mM theophylline. An additional 21.6 mg/L (0.13 mM) 1-Methylxanthine were also produced, attributed to slight activity of NdmA at the N _ 3 -position of theophylline. The 1- and 3Methylxanthine products were separated by preparative chrochromatography with less than 5 % loss during purification and were identical to commercially available standards. Purity of the isolated 3Methylxanthine was comparable to a commercially available standard, with no contaminant peaks as observed by liquid chrochromatography-mass spectrophotometry or nuclear magnetic resoresonance. Conclusions We were able to biologically produce and separate 100 mg of highly pure 3Methylxanthine from theophylline (1,3-diMethylxanthine). The N-demethylation reaction was catalyzed by E. coli engineered with N-demethylase genes, ndmA and ndmD . This microbial conversion represents a first step to develop a new biological platform for the production of Methylxanthines from economical feedstocks such as caffeine, theobromine, and theophylline.

  • Direct conversion of theophylline to 3Methylxanthine by metabolically engineered E. coli
    Microbial cell factories, 2015
    Co-Authors: Khalid H. R. Algharrawi, Ryan M. Summers, Sridhar Gopishetty, Mani Subramanian
    Abstract:

    Methylxanthines are natural and synthetic compounds found in many foods, drinks, pharmaceuticals, and cosmetics. Aside from caffeine, production of many Methylxanthines is currently performed by chemical synthesis. This process utilizes many chemicals, multiple reactions, and different reaction conditions, making it complicated, environmentally dissatisfactory, and expensive, especially for monoMethylxanthines and paraxanthine. A microbial platform could provide an economical, environmentally friendly approach to produce these chemicals in large quantities. The recently discovered genes in our laboratory from Pseudomonas putida, ndmA, ndmB, and ndmD, provide an excellent starting point for precisely engineering Escherichia coli with various gene combinations to produce specific high-value paraxanthine and 1-, 3-, and 7-Methylxanthines from any of the economical feedstocks including caffeine, theobromine or theophylline. Here, we show the first example of direct conversion of theophylline to 3Methylxanthine by a metabolically engineered strain of E. coli. Here we report the construction of E. coli strains with ndmA and ndmD, capable of producing 3Methylxanthine from exogenously fed theophylline. The strains were engineered with various dosages of the ndmA and ndmD genes, screened, and the best strain was selected for large-scale conversion of theophylline to 3Methylxanthine. Strain pDdA grown in super broth was the most efficient strain; 15 mg/mL cells produced 135 mg/L (0.81 mM) 3Methylxanthine from 1 mM theophylline. An additional 21.6 mg/L (0.13 mM) 1-Methylxanthine were also produced, attributed to slight activity of NdmA at the N 3 -position of theophylline. The 1- and 3Methylxanthine products were separated by preparative chrochromatography with less than 5 % loss during purification and were identical to commercially available standards. Purity of the isolated 3Methylxanthine was comparable to a commercially available standard, with no contaminant peaks as observed by liquid chrochromatography-mass spectrophotometry or nuclear magnetic resoresonance. We were able to biologically produce and separate 100 mg of highly pure 3Methylxanthine from theophylline (1,3-diMethylxanthine). The N-demethylation reaction was catalyzed by E. coli engineered with N-demethylase genes, ndmA and ndmD. This microbial conversion represents a first step to develop a new biological platform for the production of Methylxanthines from economical feedstocks such as caffeine, theobromine, and theophylline.

  • Novel, Highly Specific N-Demethylases Enable Bacteria To Live on Caffeine and Related Purine Alkaloids
    Journal of Bacteriology, 2012
    Co-Authors: Ryan M. Summers, Tai Man Louie, Chi-li Yu, Lokesh Gakhar, Kailin C. Louie, Mani Subramanian
    Abstract:

    ABSTRACT 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 monomononucleotide (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, 3Methylxanthine, 7-Methylxanthine, and xanthine, respectively. NdmB-His 6 plus His 6 -NdmD catalyzed N 3 -demethylation of theobromine, 3Methylxanthine, 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.

Hiroshi Ashihara – One of the best experts on this subject based on the ideXlab platform.

  • Metabolism of purine bases, nucleosides and alkaloids in theobromine-forming Theobroma cacao leaves
    Plant Physiology and Biochemistry, 2003
    Co-Authors: Yoko Koyama, Yoshihisa Tomoda, Misako Kato, Hiroshi Ashihara
    Abstract:

    Abstract 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, 14 C-labelled adenine, adenosine, guanine, guanosine, hypoxanthine and inosine were converted to salvage products (nucleotides and nucleic acids), to degradation products (ureides and CO 2 ) and to purine alkaloids (3– and 7-Methylxanthine, 7-methylxanthosine and theobromine). In contrast, 14 C-labelled xanthine and xanthosine were not used for nucleotide synthesis. They were completely degraded, but nearly 20% of [8- 14 C]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 → 3Methylxanthine → theobromine. Although no caffeine biosynthesis from 14 C-labelled purine bases and nucleosides was observed during 18 h incubations, exogenously supplied [8- 14 C]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- 14 C]Theobromine was degraded to CO 2 via 3Methylxanthine, xanthine and allantoic acid. [8- 14 C]Caffeine was catabolised to CO 2 via theophylline (1,3-diMethylxanthine) or theobromine.

  • Theophylline metabolism in higher plants.
    Biochimica et biophysica acta, 1997
    Co-Authors: Emi Ito, Alan Crozier, Hiroshi Ashihara
    Abstract:

    Abstract Metabolism of [8-14C]theophylline was investigated in leaf segments from Camellia sinensis (tea), Camellia irrawadiensis, Ilex paraguariensis (mate) and Avena sativa, root segments of Vigna mungo seedlings and cell suspension cultures of Catharanthus roseus. There was extensive uptake and metabolism of [8-14C]theophylline by leaves of tea and Camellia irrawadiensis and, to a lesser extent, mate. These purine alkaloid-containing species converted [8-14C]theophylline into 3Methylxanthine, xanthine, the ureides allantoin and allantoic acid, and CO2. With the other test systems, which were from species that do not produce purine alkaloids, there were low levels of [8-14C]theophylline uptake which were accompanied by incorporation of relatively small amounts of label into 3Methylxanthine, xanthine and CO2. None of the higher plants converted [8-14C]theophylline to either 1-methyluric acid or 1,3-dimethyluric acid, which are the main catabolites of theophylline in mammals. The data indicate that the main route of theophylline degradation in higher plants involves a theophylline→3Methylxanthine→xanthine→uric acidallantoin→allantoic acid→→CO2+NH3 pathway. In tea and mate, large amounts of [8-14C]theophylline were also converted to theobromine and caffeine via a theophylline→3Methylxanthine→theobromine→caffeine salvage pathway. The diversity of theophylline metabolism in higher plants and mammals is discussed.

  • Metabolism of Caffeine and Related Purine Alkaloids in Leaves of Tea (Camellia sinensis L.)
    Plant and Cell Physiology, 1997
    Co-Authors: Hiroshi Ashihara, Fiona M. Gillies, Alan Crozier
    Abstract:

    Purine alkaloid catabolism pathways in young, mature and aged leaves of tea (Camellia sinensis L.) were investigated by incubating leaf sections with l4 C-labelled theobromine, caffeine, theophylline and xanthine. Incorporation of label into CO2 was determined and methanolsoluble metabolites were analysed by high-performance liquid chrochromatography-radiocounting and thin layer chromatography. The data obtained demonstrate that theobromine is the immediate precursor of caffeine, which accumulates in tea leaves because its conversion to theophylline is the rate limiting step in the purine alkaloid catabolism pathway. The main fate of [8- 14 C]theophylline incubated with mature and aged leaves, and to a lesser extent young leaves, is conversion to 3Methylxanthine and onto xanthine which is degraded to 14 CO2 via the purine catabolism pathway. However, with young leaves, sizable amounts of [8- 14 C]theophylline were salvaged for the synthesis of caffeine via a 3Methylxanthine —»• theobromine —* caffeine pathway. Trace amounts of [2- 14 C]xanthine were also salvaged for caffeine biosynthesis in young leaves, by conversion to 3Methylxanthine, and this was enhanced in the presence of 5 mM allopurinol which inhibits purine catabolism. Feeds of [2- 14 C]xanthine to young leaves also indicated that 3