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." [1]

"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."[2]

• They expressed the P450s in engineered yeast strain

Caffeine to theophylline (Crozier and Ashihara)

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."[3]

Progress

"However, attempts to detect caffeine demethylase activity in the extracts in vitro are have still been (sic) unsuccessful (Ashihara and Crozier, unpublished result)." [1]

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." [2]

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."[4]

• HPLC seems to be the accepted method for quantification

References

1. Eugster HP, Probst M, Würgler FE, and Sengstag C. Caffeine, estradiol, and progesterone interact with human CYP1A1 and CYP1A2. Evidence from cDNA-directed expression in Saccharomyces cerevisiae. Drug Metab Dispos. 1993 Jan-Feb;21(1):43-9.
2. Abécassis V, Pompon D, and Truan G. High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2. Nucleic Acids Res. 2000 Oct 15;28(20):E88. DOI:10.1093/nar/28.20.e88 |
3. Ashihara H and Crozier A. Biosynthesis and catabolism of caffeine in low-caffeine-containing species of Coffea. J Agric Food Chem. 1999 Aug;47(8):3425-31. DOI:10.1021/jf981209n |
4. Georgia KA, Samanidou VF, and Papadoyannis IN. Use of novel solid-phase extraction sorbent materials for high-performance liquid chromatography quantitation of caffeine metabolism products methylxanthines and methyluric acids in samples of biological origin. J Chromatogr B Biomed Sci Appl. 2001 Aug 15;759(2):209-18. DOI:10.1016/s0378-4347(01)00251-1 |
5. Blecher R and Lingens F. The metabolism of caffeine by a Pseudomonas putida strain. Hoppe Seylers Z Physiol Chem. 1977 Jul;358(7):807-17. DOI:10.1515/bchm2.1977.358.2.807 |
6. Schwimmer S, Kurtzman RH Jr, and Heftmann E. Caffeine metabolism by Penicillium roqueforti. Arch Biochem Biophys. 1971 Nov;147(1):109-13. DOI:10.1016/0003-9861(71)90315-8 |
7. Mohapatra BR, Harris N, Nordin R, and Mazumder A. Purification and characterization of a novel caffeine oxidase from Alcaligenes species. J Biotechnol. 2006 Sep 18;125(3):319-27. DOI:10.1016/j.jbiotec.2006.03.018 |
8. Mazzafera P. Catabolism of caffeine in plants and microorganisms. Front Biosci. 2004 May 1;9:1348-59. DOI:10.2741/1339 |
9. Anaya AL, Cruz-Ortega R, and Waller GR. Metabolism and ecology of purine alkaloids. Front Biosci. 2006 Sep 1;11:2354-70. DOI:10.2741/1975 |
10. Tassaneeyakul W, Birkett DJ, McManus ME, Tassaneeyakul W, Veronese ME, Andersson T, Tukey RH, and Miners JO. Caffeine metabolism by human hepatic cytochromes P450: contributions of 1A2, 2E1 and 3A isoforms. Biochem Pharmacol. 1994 May 18;47(10):1767-76. DOI:10.1016/0006-2952(94)90304-2 |
11. Tassaneeyakul W, Mohamed Z, Birkett DJ, McManus ME, Veronese ME, Tukey RH, Quattrochi LC, Gonzalez FJ, and Miners JO. Caffeine as a probe for human cytochromes P450: validation using cDNA-expression, immunoinhibition and microsomal kinetic and inhibitor techniques. Pharmacogenetics. 1992 Aug;2(4):173-83. DOI:10.1097/00008571-199208000-00004 |
12. Bürkle L, Cedzich A, Döpke C, Stransky H, Okumoto S, Gillissen B, Kühn C, and Frommer WB. Transport of cytokinins mediated by purine transporters of the PUP family expressed in phloem, hydathodes, and pollen of Arabidopsis. Plant J. 2003 Apr;34(1):13-26. DOI:10.1046/j.1365-313x.2003.01700.x |