MetaCyc Enzyme: theobromine demethylase

Species: Pseudomonas putida No. 352

Subunit composition of theobromine demethylase = [theobromine demethylase subunit]6

The native apparent molecular mass was determined by gel filtration chromatography [Asano94].

Cloning of the gene encoding this enzyme has not been reported.

This monooxygenase was shown to be specific for theobromine demethylation at the N3 position to produce 7-methylxanthine. Based on its absorption at 415 nm and brown color in solution, it was suggested to contain either heme or nonheme iron. Pseudomonas putida No. 352 also showed high caffeine degradation activity when grown in the presence of Fe3+ [Asano94].

A soluble, NADH-dependent caffeine demethylating monooxygenase activity was also found in extracts of this organism and the formation of formaldehyde was demonstrated. In contrast to this enzyme, the caffeine demethylase activity was not inhibited by Zn2+ [Asano94].
The subunit apparent molecular mass was determined by SDS-PAGE [Asano94].

Molecular Weight of Polypeptide: 41.0 kD (experimental) [Asano94]

Molecular Weight of Multimer: 250.0 kD (experimental) [Asano94]

Gene-Reaction Schematic

Gene-Reaction Schematic

Created 06-Jul-2010 by Fulcher CA, SRI International

Enzymatic reaction of: theobromine N3-demethylase (theobromine demethylase)

Inferred from experiment

theobromine + NAD(P)H + oxygen + H+ → 7-methylxanthine + formaldehyde + NAD(P)+ + H2O

The direction shown, i.e. which substrates are on the left and right sides, is in accordance with the direction in which it was curated.

The reaction is physiologically favored in the direction shown.

In Pathways: caffeine degradation III (bacteria, via demethylation)

The assay for this enzyme activity utilized NADH. Whether or not the enzyme could utilize NADPH was not reported.

Inhibitors (Unknown Mechanism): Zn2+ [Asano94, Asano93]Kinetic Parameters:
Substrate Km (μM) Citations
theobromine 1100.0 [Asano94]


Asano93: Asano Y, Komeda T, Yamada H (1993). "Microbial production of theobromine from caffeine." Biosci. Biotech. Biochem. 57 (8) , 1286-1289.

Asano94: Asano Y, Komeda T, Yamada H (1994). "Enzymes involved in theobromine production from caffeine by Pseudomonas putida No. 352." Biosci. Biotech. Biochem., 58 (12), 2303-2304.

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Please cite the following article in publications resulting from the use of MetaCyc: Caspi et al, Nucleic Acids Research 42:D459-D471 2014
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