User:Josh K. Michener/Caffeine

Caffeine to anything other than theophylline
"Two cDNAs of human CYP1A1 and CYP1A2 were expressed in yeast Saccharomyces cerevisiae, and microsomes of transformed strains contained substantial amounts of functional heterologous enzymes. Caffeine was shown to be metabolized by CYP1A2 and CYP1A1. Both enzymes formed paraxanthine and minor amounts of theobromine; however, trimethyluric acid was exclusively formed by CYP1A1. The fact that theophylline was not formed by either enzyme anticipates the involvement of additional enzyme(s) in the primary metabolism of caffeine." eugster

"This strategy was tested using human cytochrome P450 CYP1A1 and CYP1A2 as templates.... A microtiter plate screening system was designed to achieve colorimetric detection of polycyclic hydrocarbon hydroxylation by transformed yeast cells." abecassis
 * They expressed the P450s in engineered yeast strain

Published work
"The main route in C. arabica is caffeine -> theophylline -> 3-methylxanthine -> xanthine -> uric acid -> allantoin -> allantoic acid ->-> CO2 + NH3.

In contrast, insertion of the 7Ndemethylase encoding gene from C. eugenioides into the genome of C. arabica is much more likely to produce caffeine deficiency because the eugenioides gene product will catalyze the conversion to caffeine to theophylline and the native Arabica enzymes have the capacity to rapidly degrade theophylline. Work on the isolation and characterization of this novel N-demethylase from C. eugenioides is in progress." ashihara

Progress
"However, attempts to detect caffeine demethylase activity in the extracts in vitro are have still been (sic) unsuccessful (Ashihara and Crozier, unpublished result)." 

Research goals
"Caffeine degrading bacteria, such as Pseudomonas cepacia, demethylate caffeine to yield xanthine which is further catabolised to NH3 and CO2. The pathways via which this is achieved are being investigated. Total chromosomal DNA will be isolated and used to construct a genomic library in an appropriate Escherichia coli expression vector. Clones will be cultured on minimal salt medium containing ampicillin or kanamycin, to screen for the vector, and caffeine to select for inserts that will mediate caffeine degradation. Two, isolated from positive clones, will be physically mapped and the sections of the insert encoding caffeine demethylase activity will be sequenced. C. arabica callus and suspension cultures both produce high levels of caffeine. Thus, the potential of the cloned bacterial demethylase for degradation of caffeine will be determined by transformation of C. arabica cells and studying the regulation of caffeine levels in transgenic callus and cell suspension cultures." Search for Crozier

Related note - work in a separate group
"Horn, O. (2000). "Cloning of a caffeine demethylase into a Pichia pastoris expression system."

Detection, etc.
HPLC "An automated reversed-phase high-performance liquid chromatographic (RP-HPLC) method, using a linear gradient elution, is described for the simultaneous analysis of caffeine and metabolites according to their elution order: 7-methyluric acid, 1-methyluric acid, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, 1,3-dimethyluric acid, theobromine, 1,7-dimethyluric acid, paraxanthine and theophylline." georgia
 * HPLC seems to be the accepted method for quantification