Dev, Kamal

etd@IISc Electronic Theses and Dissertations at Indian Institute of Science

5. Summary Caffeine (1,3,7-trimethylxanthme) is a plant alkaloid and a member of a class of drugs known as methylxanthines Caffeine is one of the most popular psychoactive and wdely consumed drugs in the world and IS present in the leaves, seeds and fruits of more than sixty plant specles, of which coffee, tea, kolanuts, guarana seeds, cocoa, and yebra mate are the most well known. It is consumed in numerous forms such as coffee, tea, carbonated beverages, confectionenes as well as many over-the-counter cold / flu and allergy medicines, analgesics, appetite suppressants, and stimulants However, excessive caffeine consumphon is associated with several slde effects, which has augmented the demand for decaffeinated beverages. Current methods of decaffeination employ chemical approaches, which are expensive, labor intensive and not user fnendly Consequently, biological methods of decaffeination have gamed an Impetus The transgenic technology can be used to down regulate the genes encoding enzymes responsible for caffeine biosynthesis More recently, a transgenic coffee plant was generated In wbch the gene encoding theobromine synthase was repressed by RNA interference As caffeine is anhmicrobial and pesticidal at concentrations known to be present in coffee and tea plants, such genetically modlfied caffeine-deficient transgenic coffee or tea plants would be sensitwe to pathogens Since microorganisms are more amenable to genetic mampulations, microbes have been looked upon as another alternative for chemical decaffeination Microorganisms, mostly Pseudomonas putrda, capable of degrading caffeine have been isolated from various habitats, and shown to degrade caffeine by N-demethylation However, expression of demethylase in coffee 1 tea plants is unlikely to result in caffeine-deficient plants, because these enzymes degrade caffeine into theobromme, which is the immediate precursor for caffeine biosynthesis in plants Consequently, the objectlve of the present study was to isolate a microorgamsm capable of degrading caffeine and characterize the enzyme(s) responsible for caffeine degradation Isolation of caffeine degrading microorganism and the enzyme(s) responsible for caffeine degradation: A microorganism capable of degrading caffeine as a source of carbon and nitrogen was isolated from the soil that was used to dispose coffee waste The Isolated o r g a s m  was identified as a stran of Pseudomonas putrda, based on the Bergey's manual of systematic bacteriology In order to monitor the utilization of caffelne by the organism from the growth medium, P putzda was inoculated In caffeine contalmng medlum or cornmercml coffee powder suspension Un-utilized caffelne remalnlng in the spent medlum was extracted wlth chloroform, and analyzed by reverse phase-HPLC A time dependent decrease of caffelne In the growth medium was observed The presence of a caffelne inducible caffeine hydrolyzing actlvity was observed Estimation of caffelne degradation activity In the cytosol and membrane fractions suggested that most of the activlty IS localized in the soluble fraction (cytosol) Since we have observed a caffeine inducible enzymahc actlvity m the P putrda  sola ate, our interest was to look for the presence of any caffeine- inducible protelns Comparative analysls of protein profiles of P putrda grown ~n the presence or absence of caffeine revealed the induction of two polypeptides correspond~ng to 42 and 65 kDa in the soluble fractions, when the organlsm was grown in the presence of caffeine Purification of caffeine degradation actlvity through native-PAGE fractionation led to the homogenous preparation of a 42 kDa proteln Thls suggested that caffeine Induces a 42 kDa protein for its own degradation Polyclonal antiserum was raised agalnst the pmfied 42 kDa prote~n Immunoblot analysis indicated that polyclonal antiserum could cross react wth the 42 kDa protein in the soluble extracts only when the organlsm was grown in the presence of caffeine Analytical gel filtration revealed that the enzyme responsible for caffeine degradahon exlsts as homotetramer In solution, whose monomenc size is 42 kDa Eluctdation of caffeine degradation pathway: To identify the product of caffeine degradation, assays were performed wth purlfied enzyme and the reaction product was separated on a s~lica-TLC [ ~ - m e t h ~ l - ' ~ ~ ] c a f f e ~ n e  d gradatlon product was retarded In ~ t s  rnobillty (Rf= 0 3), whereas substrate exhibited a faster mobility (Rf= 0 9). The retention and recovery of radiolabel In the caffeine degradahon product on sillca-TLC substantlate the fact that caffelne 1s not degraded by N-demethylation Spectrophotometric caffeine degradatlon assays and compmson of absolptlon maximum of punfied caffeine degradahon product m t h  xanthjne and its derivatives indicated that caffeine degradation product is different from known intermediates (theobromine, theophylline, and xanthlne) formed during demethylat~on of caffeine To elucidate the structure of caffeine degradation product, it was purified through silica column and subjected to Infrared spectroscopy and gas chromatography-mass spectrometry analyses Structural analysis showed that the caffeine degradation product is 3-amino-N-methyl-2,3-bis-methylammo acrylamide and has a mass of 158 Da Since caffelne degradabon act~vity was found to be oxygen dependent, we proposed an oxidoreductase-mediated pathway of caffeine degradation in the isolated stran of P putrda Analysis of the structure of caffeine degradation product indicated that ~t could be further degraded into three molecules of methy lane ,  which can serve as the source of carbon and nitrogen for the growth of the orgamsm Accordingly, P puhda was proficient in metabolizing methylam~ne from the growth medium Based on the catalytic mechanism, we classified the 42 kDa caffelne degrading enzyme as caffeine oxidase The proposed ~a thway of caffeine degradation in the isolated strain of Pseudomonasputlda is shown in Figure 5 1 Caffeine oxidase 3-Amino-N-methyl-2,3-bis- Caffeine methylamino-acrylamide I 0 2  c02 Lyase (?) Formaldehyde Metty famine oxiduse dehydrogenase COi4 HCHO 4 q 0  C H s N H i  4 ~ '  + 4e- Hz0 NH3 + 2 ~ '  +2e- Figure 5.1. Proposed pathway of caffeine degradation 1x1 the isolated strain of Pseudornonm outrda. Trimethyl xanth~ne (caffe~ne) 1s hydrolyzed to amno methyl b~smethyl amrnoacrylamlde by the newly Identified caffelne ox~dase, wh~ch 1s further broken down to methylamlne, poss~bly by the lyase Methylamine is metabolized to carbon diox~de and ammonla Clraracterrzation of caffeine oxidase: Klnetic analys~s howed that caffeine oxidase evh~bits a K ,  of 125 pM for caffeine, V,, of 0 3 nmol min-' and a Tli2 of 40°C Caffelne oxidase assays performed with dialyzed (aganst TE buffer) enzyme preparatlon led to a drast~c decrease in the activity In order to rescue the caffelne ox~dase activity, dlalyzed enzyme preparation was supplemented with vanous metal Ions (ca2'. M ~ ~ + ,  zn2+, cu2', co2+, ~ n * * ,  cd2*, ~ e ~ * )  In the caffeine ox~dase assays Calcium could restore -75% of the activlty, whereas none of the other metal Ions could restore the activity of this enzyme Metal chelators l ~ k e  EDTA and o- phenanthroline inhibited the enzyme activlty, thus implicating a role for metal ions in caffeine oxidase function Some of the known xanthlne oxidase inh~bitors did not affect enzyme activity, suggesting that caffeine oxidase follows a different catalyt~c mechanism than those of xanthine oxidases Inhib~t~on of caffelne ox~dase activity by coenzymes l ~ k e  NAD and FAD indicated that the enzyme 1s not a member of dehydrogenases that belong to the family of oxidoreductases Atom~c absorption spectroscopy revealed that caffeine ox~dase has bound calclum, thus ~mpllcatlng a major role for calclum Ions In caffelne oxidase funct~on Inh~b~tion of caffelne oxidase actlvity In the presence of zinc and detection of trace amount of zinc by atomlc absorption spectroscopy suggest ~ t s  structural role in caffelne oxidase funct~on Radiolabeled zlnc binding assays Indicated that both active and inactlve forms of caffeine oxidase were proficient In zinc bindlng, thus indicating a structural role for zlnc Clrcular dichroism and fluorescence measurement studies indicated that caffelne oxidase undergoes substrate (caffeine) as well as cofactor (calcium) induced conformational changes Transposon mutagenesis of gene encoding caffeerne oxidase: In order to find whether caffeine oxidase is the first enzyme in the pathway of caffelne degradation, P putzda was subjected to Tn5 mutagenesls and mutants defective in caffeine degradation were Isolated. Transposon mutagenesis was performed In such a way that the DNA adjacent to the transposon insertion could be cloned and sequenced uslng transposon specific prlmers Transposon (Tn5) was introduced Into the caffeine degrading P purida strain (In whlch rifarnycin resistance marker was Introduced in order to select the transconjugants) via conjugation Transconjugants (resistant to nfamycin and tetracycline) were screened for the loss of phenotype h c t i o n  (defect in caffeine utilization) by plat~ng on two different media, one contain~ng caffeine and other with glucose A mutant strain (1/3000) named CM751 was isolated, which dld not grow in caffeine containing medium, but grew In medium containing glucose CM751 mutant was able to transport caffeine, but defective in caffeine util~zation. Subsequent studies showed that CM75 1 mutant was defective at the first step in the pathway of caffeine degradation Activity assays, SDS-PAGE, western and total DNA Southern analysis explicitly revealed that CM751 mutant harbors a transposon insertion in the gene encoding caffeine oxidase To identify the caffeine oxidase gene, the DNA adjacent to the transposon insertion was self-cloned, transformed ~nto  DH5a s t ran  and selected for tetracycline resistance A genomic DNA clone (obtained by BgAI cleavage) of 2 8 kb was sequenced using transposon specific pnmers The sequence obtaned did not match with P putzda genome sequence However, N-terminal sequence of caffeine oxidase aligned well with the sequenced DNA Nevertheless, the deduced P putzda sequence exhibited homology with a putative oxidoreductase encoded by the megaplasmid of Sznorhzzobrum meirlofi This rases the possibility that caffeine oxidase might be encoded by an extra- chromosomal plasmid Subsequently, we could isolate a plasmid DNA of -20 kb from the strain of P putzda Compmson of plasmid preparat~ons from wild type and CM75 1 mutant indicated that transposon ~ntegrations have occurred in the plasmid. Together, the study suggests that the isolated strain of P putlda possesses a small extra-chromosomal replicon, which encodes the enzymes responsible for caffeine degradation 

A Novel Caffeine Oxidase From Pseudomonas Putida

http://etd.iisc.ac.in/static/etd/abstracts/1503/G18463-Abs.pdf

A Novel Caffeine Oxidase From Pseudomonas Putida

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etd@IISc Electronic Theses and Dissertations at Indian Institute of Science