Article pubs.acs.org/JAFC
Characterization of a Novel Resistance-Related Deoxycytidine Deaminase from Brassica oleracea var. capitata Marthandam Asokan Shibu,† Hsueh-Hui Yang,‡ Chaur-Tsuen Lo,§ Hong-Shin Lin,† Shu-Ying Liu,*,# and Kou-Cheng Peng*,† †
Department of Life Science and the Institute of Biotechnology, National Dong Hwa University, Hualien 97401, Taiwan, Republic of China ‡ Department of Medical Research, Buddhist Tzu-Chi General Hospital, Hualien 970, Taiwan, Republic of China § Department of Biotechnology, National Formosa University, Yunlin 63208, Taiwan, Republic of China # Department of Molecular Biotechnology, Da-Yeh University, Changhua 51591, Taiwan, Republic of China ABSTRACT: Brassica oleracea deoxycytidine deaminase (BoDCD), a deoxycytidine deaminase (DCD, EC 3.5.4.14) enzyme, is known to play an important role in the Trichoderma harzianum ETS 323 mediated resistance mechanism in young leaves of B. oleracea var. capitata during Rhizoctonia solani infection. BoDCD potentially neutralizes cytotoxic products of host lipoxygenase activity, and thereby BoDCD restricts the hypersensitivity-related programmed cell death induced in plants during the initial stages of infection. To determine the biochemical characteristics and to partially elucidate the designated functional properties of BoDCD, the enzyme was cloned into an Escherichia coli expression system, and its potential to neutralize the toxic analogues of 2′-deoxycytidine (dC) was examined. BoDCD transformants of E. coli cells were found to be resistant to 2′-deoxycytidine analogues at all of the concentrations tested. The BoDCD enzyme was also overexpressed as a histidine-tagged protein and purified using nickel chelating affinity chromatography. The molecular weight of BoDCD was determined to be 20.8 kDa as visualized by SDS-PAGE. The substrate specificity and other kinetic properties show that BoDCD is more active in neutralizing cytotoxic cytosine β-D-arabinofuranoside than in deaminating 2′-deoxycytinde to 2′-deoxyuridine in nucleic acids or in metabolizing cytidine to uridine. The optimal temperature and pH of the enzyme were 27 °C and 7.5. The Km and Vmax values of BoDCD were, respectively, 91.3 μM and 1.475 mM for its natural substrate 2′-deoxycytidine and 63 μM and 2.072 mM for cytosine β-D-arabinofuranoside. The phenomenon of neutralization of cytotoxic dC analogues by BoDCD is discussed in detail on the basis of enzyme biochemical properties. KEYWORDS: deoxycytidine deaminase, cytidine deaminase, cytidine analogues, nucleoside adducts
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INTRODUCTION Plant diseases caused by fungal pathogens cause adverse effects on global agricultural production. The distribution of several phytopathogenic soil fungi such as Botrytis and Rhizoctonia due to changes in farming practices has led to enormous use of chemical pesticide that also have parallel detrimental effects. Biocontrol agents have therefore been considered as an effective alternative for chemical pesticides.1 Various biocontrol agents and their mechanism of infection control are been studied extensively to find out novel means to control microbial pathogens Trichoderma harzianum ETS 323 is a fairly studied biocontrol agent known for the production of metabolites such as anthraquinones, L-amino acid oxidase and glucanases that are active against fungal pathogens.2−9 T. harzianum ETS 323 has also been previously known to mediate a novel mechanism that establishes resistance to Rhizoctonia solani infection in Brassica oleracea var. capitata plantlets.10 The plant response to a pathogenic challenge is often accompanied by rapid cell death in and around the initial infection site, a defense mechanism known as the hypersensitive response.11 This response may or may not associate with restricted pathogen growth and it represents a form of programmed cell death.12 Hypersensitivity restricts the spread © 2014 American Chemical Society
of biotrophs, but facilitates the infection by necrotrophs like R. solani.10,13 The R. solani challenge on B. oleracea leaves elevate lipoxygenase activity and causes hypersensitive tissue damage. However, leaves of plantlets that are in symbiotic association with T. harzianum ETS 323 develop resistance to R. solani infection due to transcriptional activation of resistance enzymes including deoxycytidine deaminase (DCD; EC 3.5.4.14). The induced B. oleracea deoxycytidine deaminase (BoDCD) enzyme potentially modulates the host molecular expression and neutralizes DNA adducts to maintain the DNA integrity.10 Thus, BoDCD helps B. oleracea plantlets to attenuate fatal hypersensitivity induced by R. solani and thereby enhances resistance of the host against necrotropic fungal infection. DCD enzymes are crucial enzymes in pyrimidine metabolism that catalyze the hydrolytic deamination of 2′-deoxycytidine (dC) to deoxyuridine (dU) and NH3. They are also important in cellular protection as they catalyze deamination of cytotoxic cytosine nucleoside analogs such as cytosine β-D-arabinofuranoside (cytarabine) and gemcitabine.14,15 APOBEC3A that Received: Revised: Accepted: Published: 1796
November 13, 2013 January 24, 2014 January 29, 2014 January 29, 2014 dx.doi.org/10.1021/jf4048513 | J. Agric. Food Chem. 2014, 62, 1796−1801
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E. coli cells were used for overexpression, and the transformant cells with pET28a plasmid without BoDCD insert were used as a control. One milliliter of cultured cell suspension was inoculated into a culture flask with 100 mL of LB−kanamycin medium and incubated at 37 °C at 200 rpm until the OD600 reached 0.6−0.8. The cells were induced by adding100 μL of 1 M isopropyl-β-D-thiogalactopyranoside (IPTG) and incubated at 28 °C and 150 rpm for 7 h. The cell suspension was then centrifuged at 12000g for 10 min to pellet the cells, and the pellet was washed twice with pH 7.4 phosphate-buffered saline (PBS) buffer. The cells were then suspended in 100 μL of protein extraction buffer [50 mM Tris-HCl (pH 7.4), 0.1% (v/v) Tween 20, 1 mM phenylmethanesulfonyl fluoride (PMSF), and 10% glycerol (v/v)] and sonicated on ice using the Microson XL2000 sonicator (Misonix, Farmingdale, NY, USA) at 5 W for 5 min with an intermittent 30 s interval every 30 s. The lysate was then centrifuged for 10 min at 12000g, and the soluble and insoluble proteins were respectively isolated from the supernatant and the pellet. The supernatant was loaded to a HisTrap HP 1 mL column (GE Healthcare, Little Chalfont, UK) and purified using an ATKA prime plus FPLC system (GE Healthcare) by eluting with an imidazole linear gradient of 20−500 mM. The eluted protein was desalted by a HiTrap desalting column (GE Healthcare). Purified BoDCD was visualized by Coomassie-stained 12% SDS-PAGE analysis and protein concentration determined using a Bradford Protein Assay Kit (Bio-Rad). Antibacterial Activity. The agar disk diffusion method was performed to determine the resistance exerted by the BoDCD transformants to cytarabine.19 One milliliter of culture with 1 × 107 colony-forming units (CFUs) of E. coli was plated on the Luria− Bertani agar on which filter papers of 8 mm diameter, blotted with various concentrations of 50 μL of cytarabine, were placed on the middle of the cultured plates. Kanamycin sulfate (50 μg mL−1) disks served as a positive control. After incubation at 30 °C for 24 h, the zone of inhibition around each disk was measured. Experiment was done with triplicates. DCD Assays. Enzymatic activity was determined spectrophotometrically at 27 °C by following the decrease in absorption at 282 nm.20 The reaction was performed in 300 μL of 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.2 mM substrate, and the BoDCD enzyme. One enzyme unit is defined as the amount of enzyme that catalyzes the deamination of 1 μM of dC per minute at 27 °C. Enzyme Characterization. The protein concentration was determined according to the bicinchoninic acid (BCA) method using bovine serum albumin (BSA) as the standard.21 Optimal temperature (Topt) of the purified BoDCD (1 U) was determined by measuring the activity at different temperatures (from 15 to 45 °C). To determine the optimal pH (pHopt) for the enzyme activity, the activity was measured in 0.5 M phosphate buffer, pH 5−9. Km and Vmax values of BoDCD for different substrates (dC, cytidine, and cytarbine) were determined from the initial velocities and substrate concentration. To evaluate the effects of different metal ions, the DCD activity of the purified enzyme was determined in the presence of 1 mM concentrations of different metal ions (CaCl2, MnCl2, MgCl2, HgCl2, CoCl2, CuCl2, FeCl3, KCl, Na-EDTA) in 0.5 M phosphate buffer (pH 7.5).
deaminates cytosine in single-stranded DNA are known to restrict HIV infection through deamination induced mutational inactivation.16 A similar enzyme APOBEC3C (A3C) is known to restrict herpes viruses in mammalian cells.17 APOBEC1 is known to initiate diversity in gene expression by deaminating dC to dU on single-stranded DNA and RNA.18 There are very limited reports on DCDs of terrestrial plants available so far and therefore in order to find out the prospective of their application, it is important to characterize the biochemical and functional properties of plant DCDs as studied in their mammalian counterpart. In this study, recombinant BoDCD was cloned and expressed in an Escherichia coli host system and the ability of the recombinant BoDCD to confer host resistance against cytarabine, a dC analog, was determined. The enzyme was further purified to homogeneity and characterized for their kinetic and physiological properties. Characteristics of BoDCD gives a novel insight on the functions of plant DCDs as it is the only resistance related DCD in plants reported and characterized so far.
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MATERIALS AND METHODS
All the chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless stated otherwise. Bacterial Strain and Growth Media. The E. coli Rosetta DE3 (provided by Dr. Ruey-Yi Chang, National Dong Hwa University, Taiwan) cells with recombinant and nonrecombinant PCR 2.1 plasmid were cultured in Luria−Bertani media containing 50 mg mL−1 kanamycin sulfate. Cloning of BoDCD mRNA. The BoDCD was induced in B. oleracea plants and the BoDCD cDNA was amplified as described previously.10 The primers used were 5′-GAATTCATGGAAGAAGCTAAAGTGGAAGCA-3′ (sense) and 5′-CTCGAGGTATAAACGGAACTTCTCCTTGGT-3′ (antisense). The primers were designed from the nucleotide sequence of BoDCD (GenBank accession no. AEX58668). The sense primer contained the recognition sequences for EcoRI restriction endonuclease and N-terminal region of the BoDCD, and the antisense primer contained the recognition sequences for XhoI restriction endonuclease and the coding sequences of the C-terminal region of BoDCD. The amplified cDNAs were ligated into a PCR 2.1 vector by using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA) and transferred into CaCl2-mediated competent E. coli TOP10 cells according to the manufacturer’s instructions. The positive BoDCD transformants were screened by PCR, and the PCR 2.1-BoDCD plasmids from them were isolated and sequenced by using the commercial sequencing service from Protech Technology Enterprise Co., Ltd., Taipei, Taiwan (Republic of China). Construction of Recombinant Expression Vector pET28aBoDCD. The BoDCD insert from the PCR 2.1-BoDCD vector was collected by double digestion with EcoRI and XhoI restriction enzymes. The BoDCD fragments obtained (558 + 11 bp) were analyzed with 1.5% agarose gel electrophoresis and recovered with the Qiaquick gel extraction kit (Qiagen, Valencia, CA, USA). Similarly, the expression vector pET28a (+) (5369 bp) was also digested with EcoRI and XhoI (Promega, Madison, WI, USA), and the linear plasmid was recovered and purified after separating by electrophoresis in a 1% agarose gel. The digested pET28a vector and the BoDCD insert were constructed as a pET28a-BoDCD expression vector by ligating them with T4 DNA ligase (Promega, Annandale, NSW, Australia). The recombinant expression vector construct was transformed into E. coli DH5α host, and the positive recombinant clones were further detected by PCR and verified by DNA sequencing. Induction and Expression of the Recombinant BoDCD. The recombinant BoDCD was overexpressed in E. coli Rosetta cells that provide suitable eukaryotic codons to facilitate universal translation. Specifically, screened single colonies of pET28a-BoDCD transformant
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RESULTS AND DISCUSSION Enhancement of Resistance to Cytarabine in Transformant E. coli Cells. Like the mammalian DCDs, BoDCD also can potentially convert toxic dC analogues into relatively less toxic uridine derivatives and therefore provide resistance to infection-related hypersensitivity in plants.10,22 To investigate this functional property, BoDCD was characterized for its ability to detoxify cytarabine, a cytosine base with an arabinose sugar, which is known to interfere with DNA synthesis.23,24 Cytarabine is rapidly converted to cytarabine triphosphate, which damages DNA by incorporating into the DNA during the S phase. Cytarabine also inhibits both polymerase enzymes 1797
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and nucleotide reductase enzymes that are essential for DNA synthesis.24,25 The ability of an E. coli BoDCD transformant to resist cytarabine toxicity was comparatively assessed to find the potential of the recombinant BoDCD to neutralize cytidine analogues (Figure 1). Cytarabine inhibited the growth of
Figure 1. Structures of three substrates of BoDCD: 2′-deoxycytidine (a); cytidine (b); cytarabine (c). Figure 3. SDS-PAGE analysis of the overexpressed and purified recombinant BoDCD. An aliquot of sample containing 20 μg of protein was loaded for separation. Lanes: 1, molecular weight markers; 2, total soluble proteins from bacterial lysate after overexpression; 3, purified recombinant BoDCD.
control E. coli cells at all tested concentrations. The inhibition was found to increase with increasing concentration, but the transformed colonies were found to be resistant and were protected from the cytarabine toxicity. However, at a higher concentration of 2 μM cytarabine, the BoDCD transformants showed a slight inhibition (Figure 2).
Table 1. Purification of Recombinant B. oleracea Deoxycytidine Deaminase purification step soluble lysate affinity column
total protein (mg)
total activity (U)a
specific activity (U mg−1)a
30
1639
54.6
26
1583.5
61
yield (%) 100 96.6%
a
One enzyme unit (U) is defined as the amount of enzyme that catalyzes the deamination of 1 μM 2′-deoxycytidine (dC) min−1 at 27 °C. Figure 2. BoDCD mediated resistance to cytarabine toxicity and bacteriostatic effect of cytarabine on the BoDCD transformant and the control E. coli. Error bars indicate standard error of the mean (n = 3).
functionally more active in metabolizing dC adducts such as cytarabine and comparatively less effective in deaminating dC to dU in nuclei acids and metabolizing cytidine to uridine for the pyrimidine pool. The conserved functional DCD domain (AA 27−AA 105) of BoDCD is a zinc binding region, which plays a central role in the proposed catalytic mechanism by activating a water molecule to form a hydroxide ion that performs a nucleophilic attack on the substrate. As threedimensional structures of similar DCDs have not been determined so far, the specific characteristics of the catalytic domain that are responsible for the difference in substrate specificity were not established. However, upon analysis of the structures of all three substrates used, the difference in the functional group at the 2′ carbon and their spatial configurations appear to determine the enzyme−substrate binding and activity. The Km and the Vmax of BoDCD for the substrate dC were detrermined as 91.3 μM and 1.475 mM, respectively; for cytidine the values were 368 μM and 0.599 mM, and for cytarabine the values were 63 μM and 2.072 mM (Table 2). The Km of cytidine was >3-fold higher and the Vmax was almost 1.5-fold lower than those of dC, whereas the Km of cytarabine was almost 0.45 times lower and the Vmax was 0.4-fold higher than those of dC (Table 2). On the basis of their respective kcat/Km values, the BoDCD was relatively more active when cytarabine was used as a substrate and was less active with cytidine as the substrate (Table 2). The higher BoDCD activity to cytarabine and dC can be attributed to the close structural
Catalytic Activity of the Recombinant BoDCD. To find the substrate specificity of BoDCD, the enzyme was overexpressed and purified as a C-terminal His-tagged soluble fusion protein, and its catalytic properties were determined. The size of the fusion protein (191 amino acid length) was determined as 20.8 kDa on the basis of 10% SDS-PAGE analysis (Figure 3). The theoretical mass of the native BoDCD without the His-tag was calculated as 20.01 kDa. The specific activity of BoDCD in the soluble portion of the total extract for its natural substrate dC was 54.6 U mg−1, and that of the purified enzyme (96.6% yield) was 61 U mg−1 (Table 1). Kinetic Parameters and Catalytic Properties of BoDCD. Although there are only a few reports on the characterization of plant DCDs so far, reports on plant cytidine deaminase (EC 3.5.4.5, CDA), an enzyme with a similar biochemical function, are available. The kinetic parameters of plant CDAs have been determined and reported previously. Km and Vmax of a previously known Arabidopsis thaliana CDA for dC have been reported as 49.3 and 24.4 μM, respectively.26 CDA also deaminates known cytidine analogues such as cytarabine, 5-azacytidine, and 5-methylcytidine that are formed as a product of lipoxidation and targets nucleosides and dsDNA, causing severe damage to the DNA.20 The substrate specificity and the kinetic properties show that BoDCD is 1798
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Table 2. Kinetic Parameters and Substrate Specificity of the Recombinant B. oleracea Deoxycytidine Deaminase substrate
Km (μM)
Vmax (mM min−1)
kcat/Km (μM S−1)
2′-deoxycytidine cytidine cytarabin
91.3 ± 5.6 368 ± 18.2 63 ± 12.2
1.475 ± 0.3 0.599 2.072 ± 0.2
0.342 0.034 0.697
similarity of these two substrates. The kcat/Km values of BoDCD were 0.342, 0.034, and 0.697 μM S−1 for dC, cytidine, and cytarabine, respectively. The higher affinity and higher reaction rate of BoDCD to cytarabine, according to the kinetic parameters, suggest that the enzyme binds and metabolizes cytarabine comparatively more swiftly than it binds and metabolizes cytosine. This can be also theoretically interpreted that BoDCD potentially neutralizes cytarabine in less concentration during the early stages of cytarabine accumulation but maintains a higher threshold level before dC and cytidine metabolism. Therefore, BoDCD is more sensitive and active in the detoxification mechanism than dC and cytidine metabolism. Furthermore, although BoDCD can metabolize dC and cytidine, a function contributing to DNA synthesis and expression, the fact that they are known to be induced only during three-way interaction shows that they contribute more during cytotoxic challenges and potentially provide protection by neutralizing dC analogues to protect macromolecules from adducts.10 Optimal Conditions for BoDCD Activity. Optimal conditions for the activity of plant CDAs have been determined previously, and their Topt and pHopt ranges were found to vary greatly. At-cda1, a CDA from A. thaliana, is found to be active between pH 3.0 and 10.5, beyond which the enzyme was inactive irreversibly.20 A CDA from A. thaliana was found to be active between pH 6 and 11 with a slight maximum at pH 7.5− 8. The enzyme was inactive at pH 4.27 The optimal temperature of the BoDCD was determined as 27 °C. The enzyme activity was found to be inhibited below 25 °C and above 30 °C (Figure 4). BoDCD was highly active at pH 7.5, and the activity
Figure 5. Influence of pH on the activity of the recombinant BoDCD.
This may be due to the presence of thiol groups in amino acids C90, C101, C107, C110, M112, and C113 located in the catalytic motif. All other metal ions relatively inhibited BoDCD activity by 5−30% (Table 3). Table 3. Effect of Metal Ions on the B. oleracea Deoxycytidine Deaminase Activity metal iona
relative activity (%)
metal iona
relative activity (%)
none MnCl2 CoCl2 CuCl2 CaCl2
100 70 85 15 88
EDTA KCl MgCl2 HgCl2 FeCl3
80 98 95 0 15
a
Final concentration of the various agents was 1 mM.
The biochemical and physiological functions of plant DCDs were so far not characterized and were considered as the enzymes for pyrimidine metabolism and gene expression diversity. The deduced biochemical property of BoDCD justifies previous evidence on the role of DCD in plant resistance.10 Enhancement of systemic resistance in plants to pathogens occurs due to two different mechanisms. The systemic acquired resistance (SAR) is mediated by a salicylic acid-dependent pathway and involves the role of pathogenesisrelated proteins. SAR develops either locally or systemically according to the pathogen challenged, as a result of a successful infection or a hypersensitive response. The induced systemic resistance (ISR) is a response to colonization of plant roots by certain rhizosphere microorganisms. They are mediated by jasmonic acid/ethylene pathways. The biocontrol agent, Trichoderma, is commonly known as an inducer of ISR by root colonization.29 As BoDCD is an enzyme induced during three-way interaction between B. oleracea, T. harzianum ETS 323, and R. solani that also involves hypersensitivity, the possibility of BoDCD being a cross talk between SAR and ISR is evident. The functional properties of BoDCD, apart from revealing the antimicrobial potential of BoDCD, show that BoDCD can be used in other possible applications such as in ameliorating the adverse effect of dC analogues during chemotherapy. Normal intestinal florae help to maintain intestinal structure, function, and defense. Cytotoxic chemotherapeutic drugs such as cytarabine are detrimental to such protective florae. When the ecological balance of the intestine is damaged, a lot of bacteria and endotoxin enter into the circulation by the portal system and cause intestinal endotoxemia; under certain
Figure 4. Influence of temperature on the activity of the recombinant BoDCD.
dropped significantly beyond this range (Figure 5). As the normal pH in the cytoplasm of most higher plants is slightly alkaline (7.4−7.5), the pHopt of BoDCD suggests that they are potentially active during normal cytoplasm conditions without proton fluxes.28 The ability of dC deamination by BoDCD was strongly inhibited in the presence of 1 mM Hg2+ and substantially inhibited in the presence of Cu2+ and Fe3+ ions. 1799
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derived from Trichoderma harzianum ETS 323. FEBS J. 2011, 278, 3381−3394. (10) Shibu, M. A.; Lin, H. S.; Yang, H. H.; Peng, K. C. Trichoderma harzianum ETS 323-mediated resistance in Brassica oleracea var. capitata to Rhizoctonia solani involves the novel expression of a glutathione S-transferase and a deoxycytidine deaminase. J. Agric. Food Chem. 2012, 60, 10723−10732. (11) Lam, E.; Kato, N.; Lawton, M. Programmed cell death, mitochondria and the plant hypersensitive response. Nature 2001, 411, 848−853. (12) Heath, M. C. Hypersensitive response-related death. Plant Mol. Biol. 2000, 44, 321−334. (13) Govrin, E. M.; Levine, A. The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea. Curr. Biol.: CB 2000, 10, 751−757. (14) Mancini, W. R. The role of deoxycytidine-metabolizing enzymes in the cytotoxicity induced by 3′-amino-2′,3′-dideoxycytidine and cytosine arabinoside. Cancer Chemother. Pharmacol. 1992, 30, 139− 144. (15) Sigmond, J.; Honeywell, R. J.; Postma, T. J.; Dirven, C. M. F.; de Lange, S. M.; van der Born, K.; Laan, A. C.; Baayen, J. C. A.; Van Groeningen, C. J.; Bergman, A. M.; Giaccone, G.; Peters, G. J. Gemcitabine uptake in glioblastoma multiforme: potential as a radiosensitizer. Ann. Oncol. 2009, 20, 182−187. (16) Love, R. P.; Xu, H.; Chelico, L. Biochemical analysis of hypermutation by the deoxycytidine deaminase APOBEC3A. J. Biol. Chem. 2012, 287, 30812−30822. (17) Suspene, R.; Aynaud, M. M.; Koch, S.; Pasdeloup, D.; Labetoulle, M.; Gaertner, B.; Vartanian, J. P.; Meyerhans, A.; WainHobson, S. Genetic editing of herpes simplex virus 1 and Epstein-Barr herpesvirus genomes by human APOBEC3 cytidine deaminases in culture and in vivo. J. Virol. 2011, 85, 7594−7602. (18) Conticello, S. G. The AID/APOBEC family of nucleic acid mutators. Genome Biol. 2008, 9, 229. (19) Almeida, A. A.; Farah, A.; Silva, D. A.; Nunan, E. A.; Gloria, M. B. Antibacterial activity of coffee extracts and selected coffee chemical compounds against enterobacteria. J. Agric. Food Chem. 2006, 54, 8738−8743. (20) Vincenzetti, S.; Cambi, A.; Neuhard, J.; Schnorr, K.; Grelloni, M.; Vita, A. Cloning, expression, and purification of cytidine deaminase from Arabidopsis thaliana. Protein Express. Purif. 1999, 15, 8−15. (21) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150, 76−85. (22) Letourneau, S.; Palerme, J. S.; Delisle, J. S.; Beausejour, C. M.; Momparler, R. L.; Cournoyer, D. Coexpression of rat glutathione Stransferase A3 and human cytidine deaminase by a bicistronic retroviral vector confers in vitro resistance to nitrogen mustards and cytosine arabinoside in murine fibroblasts. Cancer Gene Ther. 2000, 7, 757−765. (23) Kufe, D. W.; Major, P. P. Studies on the mechanism of action of cytosine arabinoside. Med. Pediatr. Oncol. 1982, 10 (Suppl. 1), 49−67. (24) Carbone, G. M.; Catapano, C. V.; Fernandes, D. J. Imbalanced DNA synthesis induced by cytosine arabinoside and fludarabine in human leukemia cells. Biochem. Pharmacol. 2001, 62, 101−110. (25) Perry, M. C. The Chemotherapy Source Book, 4th ed.; Wolters Kluwer Health/Lippincott Williams & Wilkins: Philadelphia, PA, USA, 2008; p xvi, 779 pp. (26) Kafer, C.; Thornburg, R. Arabidopsis thaliana cytidine deaminase 1 shows more similarity to prokaryotic enzymes than to eukaryotic enzymes. J. Plant Biol. 2000, 43, 162−170. (27) Faivre-Nitschke, S. E.; Grienenberger, J. M.; Gualberto, J. M. A prokaryotic-type cytidine deaminase from Arabidopsis thaliana gene expression and functional characterization. Eur. J. Biochem./FEBS 1999, 263, 896−903.
conditions it can also stimulate a chain reaction of the cytokines and other inflammatory mediators and cause systemic organ damage.30−34 The DCD enzymes can be effectively used to neutralize the side effects of cytotoxic cytidine adducts that are employed as drugs. As the affinity of BoDCD to cytarabine is greater than to its natural substrate DCD, the enzyme can be potentially considered for providing resistance to intestinal florae against cytidine analogues. Engineered microbes with BoDCD expression can be used as probiotics to counter the effects of cytarabine.35 Further exploration on BoDCD will reveal interesting insights and applications.
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AUTHOR INFORMATION
Corresponding Author
*(K.-C.P.) Phone: +886 3 863 3635. Fax: +886 3 863 3630. Email:
[email protected]. (S.-Y.L.) Phone: 886 4 8511 888 ext. 4256. Fax: 886 4 8511 326. E-mail:
[email protected]. edu.tw. Funding
This work was partly supported by grants from the National Science Council, Taiwan (NSC 99-2313-B-212-001-MY3 and NSC 102-2313-B-259 -003). This work is part of the Ph.D. thesis of M. A. Shibu, who was financially supported by the Taiwan scholarship awarded by the Ministry of Foreign Affairs of the Republic of China and from the Department of Life Science, National Dong Hwa University. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank B. Y. Huang, H. N. Shih, and F. J. Tsai from K.-C. Peng’s laboratory for all their help that facilitated this work.
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REFERENCES
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Journal of Agricultural and Food Chemistry
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dx.doi.org/10.1021/jf4048513 | J. Agric. Food Chem. 2014, 62, 1796−1801
