Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex

Kim, Jun Hoe; Kim, Bong Heon; Brooks, Shelby M.; Kang, Shin-Gak; Summers, Ryan M.; Song, Hyun Kyu

doi:10.1016/j.jmb.2019.08.004

Cited by 39 publications

(63 citation statements)

References 46 publications

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“…Indeed, although we find that these enzymes share 88-percent sequence identity with one another, the 195-215 loop is only 52-percent identical in sequence and only 29% identical over the portion (201-214) that plugs the active site tunnel in SxtT. Different orientations of the equivalent loop have been observed in dicamba monooxygenase 48 and NdmA/NdmB 37 and, similar to what we propose for SxtT and GxtA, have been suggested to either permit or restrict access of substrate to the active site and/or promote product release. Consistent with this hypothesis, GxtA (which showcases the "open" conformation) exhibits a broader substrate scope than SxtT 16 .…”

Section: Discussionmentioning

confidence: 66%

“…1a (light blue) and catalyze the oxidative degradation of aromatic compounds (IPR001663, IPR015879). The remaining seven structurally elucidated Rieske oxygenases are also involved in degradative pathways: carbazole 1,9α-dioxygenase 33 , 2-oxoquinoline 8-monooxygenase 34 , dicamba monooxygenase 35,36 , stachydrine demethylase (2ZYL), and NdmA/ NdmB 37 . These enzymes each degrade aromatic heterocycles and/ or phenols, whereas 3-ketosteroid-9-α-hydroxylase (KshA 15 ) catabolizes the cycloalkane rings of ketosteroids.…”

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confidence: 99%

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Structural basis for divergent C–H hydroxylation selectivity in two Rieske oxygenases

et al. 2020

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Biocatalysts that perform C-H hydroxylation exhibit exceptional substrate specificity and site-selectivity, often through the use of high valent oxidants to activate these inert bonds. Rieske oxygenases are examples of enzymes with the ability to perform precise mono-or dioxygenation reactions on a variety of substrates. Understanding the structural features of Rieske oxygenases responsible for control over selectivity is essential to enable the development of this class of enzymes for biocatalytic applications. Decades of research has illuminated the critical features common to Rieske oxygenases, however, structural information for enzymes that functionalize diverse scaffolds is limited. Here, we report the structures of two Rieske monooxygenases involved in the biosynthesis of paralytic shellfish toxins (PSTs), SxtT and GxtA, adding to the short list of structurally characterized Rieske oxygenases. Based on these structures, substrate-bound structures, and mutagenesis experiments, we implicate specific residues in substrate positioning and the divergent reaction selectivity observed in these two enzymes.

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Section: Discussionmentioning

confidence: 66%

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confidence: 99%

Structural basis for divergent C–H hydroxylation selectivity in two Rieske oxygenases

et al. 2020

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“…This activity could reflect a natural promiscuity with respect to substrate for this set of enzymes, or it could be the result of an artificial characteristic of our system. NdmA and NdmB normally function as an A 3 :B 3 heterocomplex of trimers when both are expressed in the same bacterial cell (25). A reverse binding configuration in which the xanthine ring is flipped in the substrate binding site between the two a Averages and 95% confidence intervals (95% CI) and are reported from three measurements.…”

Section: Discussionmentioning

confidence: 99%

“…The genes encode three distinct N-demethylases (NdmA, NdmB, and NdmC), an accessory protein (NdmD), and an NdmE homologue (22,24). NdmA and NdmB normally associate to form a heterocomplex, and they, respectively, remove the N 1 and N 3 methyl groups of caffeine and other methylxanthines (25). NdmC demethylates the N 7 position.…”

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confidence: 99%

Bioassay for Determining the Concentrations of Caffeine and Individual Methylxanthines in Complex Samples

Gutierrez

Shah

Rex

et al. 2019

Appl Environ Microbiol

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Caffeine and other methylxanthines are stimulant molecules found in formulated beverages, including sodas and energy drinks, and in brewed beverages, such as coffee and teas. Previously, we developed a bioassay for caffeine that involves monitoring the growth of a ΔguaB mutant of Escherichia coli defective in de novo guanine biosynthesis. When supplemented with a plasmid expressing the genes for an N-demethylation pathway from Pseudomonas putida CBB5, these bacteria demethylate caffeine (1,3,7-trimethylxanthine) and other methylxanthines into xanthine, which is then converted into guanine to support cell growth. A major limitation of this bioassay was that it could only measure the total concentration of all methylxanthines in a mixture. Therefore, it could not be used to measure the caffeine content of beverages like teas, which contain substantial quantities of multiple methylxanthines. To overcome this limitation, we created seven new plasmids containing all subsets of the three demethylase genes (ndmA, ndmB, and ndmC). We show that strains of ΔguaB E. coli containing each plasmid are able to demethylate specific subsets of methylxanthines and that they can be used to determine the concentrations of individual methylxanthines in complex mixtures containing multiple methylxanthines, including coffee doped with an additional methylxanthine. While validating this assay, we also discovered an unexpected demethylation event at the 1-methyl position when NdmB and NdmC were expressed in the absence of NdmA. The improved cell-based bioassay is inexpensive, is easy to use, and gives results comparable to standard high-performance liquid chromatography methods for measuring methylxanthine concentrations. IMPORTANCE Caffeine (1,3,7-trimethylxanthine) is the dominant neurostimulant found in coffee, teas, sodas, and energy drinks. Measuring the amount of caffeine and other methylxanthines in these beverages is important for quality assurance and safety in food science. Methylxanthines are also used in medicine and as performance-enhancing drugs, two contexts in which accurately determining their concentrations in bodily fluids is important. Liquid chromatography is the standard method for measuring methylxanthine concentrations in a sample, but it requires specialized equipment and expertise. We improved a previous bioassay that links E. coli growth to methylxanthine demethylation so that it can now be used to determine the amounts of individual methylxanthines in complex mixtures or beverages, such as coffee.

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“…Curiously, none of the strains with only a subset of genes contain ndmB homologs. However, given that as few as two mutations to the ndmA active site are enough to change the activity from N 1 -to N 3 -demethylation (Kim et al, 2019) some of the genes identified as ndmA may have a broader positional specificity than those which have already been characterized. Alternatively, there may be unculturable bacteria with ndmB homologs that we were not able to detect using the approach described here.…”

Section: Stenotrophomonas Maltophiliamentioning

confidence: 99%

Cultivation and Genome Sequencing of Bacteria Isolated From the Coffee Berry Borer (Hypothenemus hampei), With Emphasis on the Role of Caffeine Degradation

et al. 2021

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The coffee berry borer, the most economically important insect pest of coffee worldwide, is the only insect capable of feeding and reproducing solely on the coffee seed, a food source containing the purine alkaloid caffeine. Twenty-one bacterial species associated with coffee berry borers from Hawai’i, Mexico, or a laboratory colony in Maryland (Acinetobacter sp. S40, S54, S55, Bacillus aryabhattai, Delftia lacustris, Erwinia sp. S38, S43, S63, Klebsiella oxytoca, Ochrobactrum sp. S45, S46, Pantoea sp. S61, Pseudomonas aeruginosa, P. parafulva, and Pseudomonas sp. S30, S31, S32, S37, S44, S60, S75) were found to have at least one of five caffeine N-demethylation genes (ndmA, ndmB, ndmC, ndmD, ndmE), with Pseudomonas spp. S31, S32, S37, S60 and P. parafulva having the full complement of these genes. Some of the bacteria carrying the ndm genes were detected in eggs, suggesting possible vertical transmission, while presence of caffeine-degrading bacteria in frass, e.g., P. parafulva (ndmABCDE) and Bacillus aryabhattai (ndmA) could result in horizontal transmission to all insect life stages. Thirty-five bacterial species associated with the insect (Acinetobacter sp. S40, S54, S55, B. aryabhattai, B. cereus group, Bacillus sp. S29, S70, S71, S72, S73, D. lacustris, Erwinia sp. S38, S43, S59, S63, K. oxytoca, Kosakonia cowanii, Ochrobactrum sp. S45, S46, Paenibacillus sp. S28, Pantoea sp. S61, S62, P. aeruginosa, P. parafulva, Pseudomonas sp. S30, S31, S32, S37, S44, S60, S75, Stenotrophomonas sp. S39, S41, S48, S49) might contribute to caffeine breakdown using the C-8 oxidation pathway, based on presence of genes required for this pathway. It is possible that caffeine-degrading bacteria associated with the coffee berry borer originated as epiphytes and endophytes in the coffee plant microbiota.

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Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex

Cited by 39 publications

References 46 publications

Structural basis for divergent C–H hydroxylation selectivity in two Rieske oxygenases

Structural basis for divergent C–H hydroxylation selectivity in two Rieske oxygenases

Bioassay for Determining the Concentrations of Caffeine and Individual Methylxanthines in Complex Samples

Cultivation and Genome Sequencing of Bacteria Isolated From the Coffee Berry Borer (Hypothenemus hampei), With Emphasis on the Role of Caffeine Degradation

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