Characterization of a broad-specificity non-haem iron N-demethylase from Pseudomonas putida CBB5 capable of utilizing several purine alkaloids as sole carbon and nitrogen source.
TLDR
Ndm was deduced to be a Rieske [2Fe-2S]-domain-containing non-haem iron oxygenase based on its distinct absorption spectrum and significant identity of the N-terminal sequences of NdmA and NdmB with the gene product of an uncharacterized caffeine demethylase in P. putida IF-3 and a hypothetical protein in Janthinobacterium sp.Abstract:
N-Demethylation of many xenobiotics and naturally occurring purine alkaloids such as caffeine and theobromine is primarily catalysed in higher organisms, ranging from fungi to mammals, by the well-studied membrane-associated cytochrome P450s. In contrast, there is no well-characterized enzyme for N-demethylation of purine alkaloids from bacteria, despite several reports on their utilization as sole source of carbon and nitrogen. Here, we provide what we believe to be the first detailed characterization of a purified N-demethylase from Pseudomonas putida CBB5. The soluble N-demethylase holoenzyme is composed of two components, a reductase component with cytochrome c reductase activity (Ccr) and a two-subunit N-demethylase component (Ndm). Ndm, with a native molecular mass of 240 kDa, is composed of NdmA (40 kDa) and NdmB (35 kDa). Ccr transfers reducing equivalents from NAD(P)H to Ndm, which catalyses an oxygen-dependent N-demethylation of methylxanthines to xanthine, formaldehyde and water. Paraxanthine and 7-methylxanthine were determined to be the best substrates, with apparent K
m and kcat
values of 50.4±6.8 μM and 16.2±0.6 min−1, and 63.8±7.5 μM and 94.8±3.0 min−1, respectively. Ndm also displayed activity towards caffeine, theobromine, theophylline and 3-methylxanthine, all of which are growth substrates for this organism. Ndm was deduced to be a Rieske [2Fe–2S]-domain-containing non-haem iron oxygenase based on (i) its distinct absorption spectrum and (ii) significant identity of the N-terminal sequences of NdmA and NdmB with the gene product of an uncharacterized caffeine demethylase in P. putida IF-3 and a hypothetical protein in Janthinobacterium sp. Marseille, both predicted to be Rieske non-haem iron oxygenases.read more
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Journal ArticleDOI
Novel, Highly Specific N-Demethylases Enable Bacteria To Live on Caffeine and Related Purine Alkaloids
TL;DR: This work reports the first report of bacterial N-demethylase genes that enable bacteria to live on caffeine and represent a new class of Rieske oxygenases and have the potential to produce biofuels, animal feed, and pharmaceuticals from coffee and tea waste.
Journal ArticleDOI
Micropollutant degradation via extracted native enzymes from activated sludge
TL;DR: The approach presented in this study provides the experimental basis for a further elucidation of the enzymatic processes underlying wastewater treatment on the level of native proteins.
Journal ArticleDOI
Genetic characterization of caffeine degradation by bacteria and its potential applications.
TL;DR: Various biotechnological applications of these genes responsible for bacterial caffeine degradation, including bio‐decaffeination, remediation of caffeine‐contaminated environments, production of chemical and fuels and development of diagnostic tests have also been demonstrated.
Journal ArticleDOI
Caffeine degradation by methanogenesis: Efficiency in anaerobic membrane bioreactor and analysis of kinetic behavior
TL;DR: A reaction equation for the caffeine degradation by methanogenesis based on the stoichiometric relationship between the degraded caffeine and the increased end-products: CH4, NH4+-N and CO2 is proposed and confirmed and provided a new thought to microbial caffeine degradation.
Book ChapterDOI
Xanthine Alkaloids: Occurrence, Biosynthesis, and Function in Plants.
TL;DR: The in planta role of caffeine in chemical defense that has been demonstrated using transgenic caffeine-forming tobacco and chrysanthemum plants, which are resistant to attack by pathogens and herbivores are considered.
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