Nucleoside Degradation in Some Streptomyces Strains
Nadia H. Ali
Latifa A. Mohamed
Five strains of Streptomyces were screened for the abilities of
their extracts to catalyze the hydrolytic or deaminating activities of
purine and pyrimidine ribonucleosides and their bases. These studies are
rare in Streptomyces. No hydrolytic cleavage for N-glycosidic
bond of nucleosides was observed in all screened strains. Hydrolytic deamination
was the only degradative activity occurred with cytidine (as substrate)
from the ribonucleosides and their bases tested. Streptomyces hygroscopicus
NRRL B-1476 gave the highest level of the hydrolytic deamination of cytidine
to uridine. Uridine was chromatographically identified in cell-free extracts.
Optimum pH and temperature of the enzyme activity were determined at 7.0
and 50Â°C, respectively. Thermal stability experiments indicated that
the enzyme completely restored its activity at 50Â°C for 30 min, however
a complete loss in enzyme activity was recorded when the enzyme was incubated
at 80 and 90Â°C for 20 and 5 min, respectively. Dialyzed extract caused
an increase in enzyme activity of about 55%. Results obtained indicate
the involvement of sulfhydryl group(s) in the catalytic site of the enzyme.
HgCl2, CuSO4 and FeCl3 (10-2
M) caused a complete inhibition of enzyme activity, whereas, little enzyme
activity was retained in presence of AgCl2, MgSO4,
BaCl2 and NaCl. Inhibition by uridine was of the competitive
type and the enzyme exhibited classic Michaelis Menten saturation kinetics.
Its apparent Km and Ki values were found to be 4.16
and 21.9 mM, respectively.
Cytidine deaminase (CDA; EC 22.214.171.124) is an enzyme of the pyrimidine
salvage pathways catalyzing the hydrolytic deamination of cytidine and
deoxycytidine to the corresponding uridine and deoxyuridine. In bacteria,
the enzyme is apparently involved in the salvage pathway of pyrimidines;
the catabolic breakdown and recycling of nucleoside compounds. In higher
eukaryotes the role and regulation of CDA is less well defined, but it
has been extensively studied due to its ability to inactivate several
important cytidine-based anticarcinogenic drugs, such as cytosine arabinoside
and 5-azacytidine (Emmanuelle et al., 1999). Cytidine deaminase
(CDA) purified from human placenta revealed the presence of five isoenzymatic
forms that differ only in their isoelectric point. (Vincenzetti et
al., 2004). The clinical interest of the human enzyme is due to its
capability to deaminate several antitumoral and antiviral cytosine nucleoside
analogs, such as the anti-leukemic agent 1-Î²-D-arabinofuranosylcytosine
and the anti-cancer agent 50-azadeoxycytidine, leading to their pharmacological
inactivation (De Clerq, 2001). Activation-induced cytidine deaminase (AID)
is required for Ig class switch recombination, a process that introduces
DNA double-strand breaks in B cells. AID catalyzes cytidine deamination
that originates DNA double-strand breaks needed for recombination and
it promotes DNA damage response and cell survival (Wu et al., 2005;
Krause et al., 2006). The enzymatic mechanisms of deaminating cleavage
of nucleosides were developed largely through studies on mammalian tissues,
bacteria, yeast and fungi. Cytidine deaminase has been reported from different
filamentous fungi namely Aspergillus fumigatus IFO 5840 (Kim and
Ha, 1992), A. niger (Elzainy et al., 1989), A. niger
NRRL3 (Ali, 1998), A. terricola (Mohamed, 1993), Penicillium
citrinum (Allam et al., 1991), P. sizowi (Bezborodov
et al., 1974). Ali and Elzainy (2000) reported that extracts of Aspergillus
niger NRRL3 catalyzed the hydrolytic deamination of only cytidine
from the tested ribonucleotides and ribonucleosides and bases, on the
other hand do not catalyzed the cleavage of N-glycosidic linkage of AMP,
GMP and UMP and their corresponding nucleosides. Recently Elshafei et
al. (2005) found that extracts of Penicillium politans NRC-510
could catalyze the deamination of cytidine to uridine maximally at pH
6.5 and 80Â°C. Concerning the purification of enzyme many attempts
have been made by many authors to purify and characterize cytidine deaminase
from Bacillus subtilis ED213 (Park et al., 1994) and Arabidopsis
thaliana (Vincenzetti et al., 1999). Pyrimidine base supplementation
to the culture medium was found to increase curdlan production by Agrobacterium
sp. ATCC 31749 (West, 2006). As far as the authors are aware similar
studies were reported only by (Elawarmy and Elzainy, 1985), they studied
deamination of cytidine and cytosine in cell-free extracts of Streptomyces
viridiviolaceus, so the aim of the study presented was to examine
the presence of the enzyme(s) responsible for the degradation of purine
and pyrimidine ribonucleosides in some other Streptomyces strains.
MATERIALS AND METHODS
Five Streptomyces strains were used throughout this study,
namely Str. erythrus NRRL ISP 5517; Str. erythrus NRRL 2338;
Str. griseus NRRL B-2682 and Str. hygroscopicus NRRL B-1476.
These cultures were obtained from Northern Utilization Research and Development
Division, US Department of Agriculture, Peoria, Illinois, USA) and Str.
sp. NRC11 was obtained from the Department of Microbial Chemistry, National
Research Center, El-Tahrir Street, Dokki, Cairo, Egypt.
Organisms were maintained on solid Czapek-Dox`s medium. The following
liquid medium was used throughout this study (g 100 mL-1):
glucose 3.0 NaNO3 0.2, K2 HPO4 0.1, MgSO4
7H2O 0.05 and KCl 0.05. The pH value was adjusted at 6.0 before
Cultivation of the Organisms
Spores were scraped and suspended in sterile distilled water. Equal
portions of spore suspensions were prepared to inoculate, under aseptic
conditions, 250 mL Erlenmeyer flasks, each containing 50 mL of sterile
medium. The inoculated flasks were then incubated statically at 28Â°C
for 4 days.
Preparation of Cell-Free Extracts
Mycelia were ground with twice their weight of washed cold sand in
a cold mortar. The obtained slurry was centrifuged at 5500 rpm for 10
min and the supernatant was used as the crude enzyme preparation.
Dialysis of Extracts
Dialysis of the extracts was made against 200 volumes of cold 0.02M Tris-HCl
buffer pH 7.0 for 2 h at 7Â°C using dialysis bags (sacks). Dialysis
sacks (cellulose tubing 21 mm dia.) were obtained from SIGMA Diagnostic,
St Louis, MO 631 78 USA.
Ribose was determined by the method of Ashwell (1957). Ammonia was estimated
by Nessler`s reagent. Protein was determined by the method of Sutherland
et al. (1949).
Enzyme Activity Determination
Purine and pyrimidine ribonucleoside hydrolase was estimated by measuring
the ribose formed from the nucleoside by the enzyme action. Aminohydrolase
was assayed by measuring the amount of ammonia which appeared after incubation
of nucleobases and their nucleosides with the enzyme. This was accompanied
by chromatographic identification of the products formed.
Identification of Pyrimidine Nucleoside
Chromatographic identification of the pyrimidine nucleoside was made using
chromatographic Whatman No. 3 MM filter paper and two solvent systems.
Solvent 1: n-butanol-glacial acetic acid-water (120: 30: 50) and solvent
2: n-butanol-formic acid-water (154: 20: 26) (Simth and Seakins, 1976).
The spots were located with an ultraviolet lamp (VL 215 LC, Vilber Lourmat,
Screening of Streptomyces Strains
Five strains of Streptomyces (Str. erythrus NRRL ISP
5517; Str. erythrus NRRL 2338; Str. griseus NRRL B-2682;
Str. sp. NRC 11 and Str. hygroscopicus NRRL B-1476) were
screened for the abilities of their extracts to catalyze the hydrolytic
and or the deaminating activities towards some purine and pyrimidine nucleosides
and bases. Ribose was not detected in the two sets of reaction mixtures
(one set contained purine ribonucleosides, adenosine, guanosine or inosine
and the other set contained pyrimidine ribonucleosides cytidine, uridine
and pyrimidine deoxy ribonucleoside, thymidine plus the fresh extracts
(at pH 4.0, 6.0 and 8.0) of five Streptomyces under study indicating
the absence of purine ribonucleoside and pyrimidine ribonucleoside and
pyrimidine deoxyribonucleoside hydrolase in these extracts (Table 1),
whereas ammonia was detected only in the reaction mixtures that contained
cytidine plus extracts of. Str. erythrus NRRL 2338; Str. sp.
NRC11 and Str. Hygroscopicus NRRL B-1476 at the same pH values
and at the same experimental conditions. On the other hand Str.griseus
NRRL B-2682 and Str. erythrus NRRRL ISP5517 could not catalyze
the hydrolytic deamination of any nucleosides or their bases (Table 1).
On comparing the levels of cytidine deaminase activity in the previously
mentioned five Streptomyces strains, it is noted that the enzyme
was found in a relatively high amounts in extracts of Str. hygroscopicus
NRRL B-1476 as compared with other two organisms (Str. erythrus
NRRL 2338; Str. sp. NRC11) so it was selected for further studies.
||Occurrence of deaminating activities in reaction mixtures containing
nucleosides or their bases at different pH values in extracts of some
|Reaction mixture contained: nucleoside or base, 5 Î¼moles;
citrate buffer or Tris-HCl, 100 Î¼moles; extract protein, 1.2
mg; total volume, 1 mL; temperature, 40Â°C and reaction time, 60
||Cytidine deaminase as a function of time of the reaction by extracts
of Str. hygroscopicus. Reaction mixture contained: cytidine
15 Î¼moles; phosphate buffer (pH 6.0), 300 Î¼moles; extract
protein 1.9 mg; total volume, 3 mL; temperature, 40Â°C and reaction
time as indicated
Chromatographic Identification of the Products
Ammonia was chromatographically identified in reaction mixture that contained
cytidine and the fresh cell-free extracts. The developed spots of the
identified and authentic uridine had the same Rf values of
0.4 in solvent 1 and 0.25 in solvent 2. The results obtained indicate
the presence of hydrolytic deaminating activity in extracts of Str.
hygroscopicus NRRL B-1476.
Time of Reaction
Reaction mixtures were incubated for 180 min at 40Â°C at different
incubation periods and after different time intervals aliquots were removed
for ammonia determination. Figure 1 shows that there
is a gradual increase in the amounts of ammonia formed by cytidine deaminase
up to 150 min of incubation, after that there is a steady state occurred
in which the increase in incubation time does not reflect noticeable increase
in the amounts of ammonia formed from cytidine.
To determine precisely the optimum pH value(s) at which optimum deamination
of cytidine by cell-free extracts of Str. hygroscopicus NRRL B-1476,
occurred, a reaction mixtures were made each contained the same amount
of protein, the same amount of cytidine and the same amount of buffer.
Citrate-phosphate and phosphate buffers were adjusted at pH 3.0 to 8.0.
The reaction mixtures were then incubated at 40Â°C for 60 min after
which determination of ammonia was carried out in all of them. Figure
2 shows that extracts of the experimental Str. hygroscopicus
NRRL B-1476 strain could catalyze ammonia release from ribonucleoside
cytidine over a wide range of pH values. Optimum pH value was recorded
at pH 7.0 for the enzymatic deamination of cytidine, while as, enzyme
activity at pH 4.0 and 8.0 were recorded about 50 and 83%, respectively
after 60 min of incubation.
||Cytidine deaminase activity as a function of pH value. Reaction
mixture contained: cytidine 5.0 Î¼moles; buffer as indicated,
100 Î¼moles; extract protein 0.36 mg; total volume, 1 mL; temperature,
40Â°C and reaction time 60 min
||Activity for cytidine of Str. hygroscopicus as a function
of temperature. Reaction mixture contained: cytidine 5.0 Î¼moles;
phosphate buffer (pH 6.0), 100 Î¼moles; extract protein 0.36 mg;
total volume, 1 mL; temperature, as indicated and reaction time 60
Temperature Dependence of Cytidine Deaminase Activity
Reaction mixtures containing cytidine as substrate were incubated at different
degrees of temperature ranged from 30-90Â°C for 60 min. Results obtained
in Fig. 3 indicated that optimum temperature was obtained
at 50Â°C and a considerable amount of enzyme activity was recorded
at 40Â°C (52%) and 60Â°C (83.6%) as compared with that obtained
at 50Â°C. Increasing the temperature above 50Â°C resulted to a gradual
decrease in enzyme activity. On the other hand, incubating the enzyme
at 80 and 90Â°C caused loss of cytidine deaminase activity (41.5 and
||Thermal stability behavior of cytidine deaminase by extracts of
Str. hygroscopicus. Reaction mixture contained: cytidine 5.0
Î¼moles; Tris-HCl buffer (pH 7.0), 100 Î¼moles; extract protein
1.1 mg; total volume, 1 mL; temperature 50Â°C and reaction time
||Effect of different buffer systems on cytidine ribonucleoside deaminase
activity of Str. hygroscopicus NRRL B-1476
|Reaction mixture contained: Cytidine, 5 Î¼moles;
Buffer (pH 7.0), 100 Î¼moles; Extract protein, 1.43 mg; Total
volume, 1 mL; Temperature, 50Â°C, Reaction time, 60 min
Heat Inactivation Kinetics
To test the stability of the enzyme as a function of exposure to 50-90Â°C
in absence of the substrate, aliquots of the exposed crude enzyme preparations
were withdrawn at different time intervals, cooled then incubated and
assayed for cytidine deaminase activity. Results obtained are graphically
represented in Fig. 4, which shows a complete stability
of the enzyme activity when incubated at 50Â°C for 30 min. Gradual
inactivation were noticed when the enzyme was incubated at 60 and 70Â°C,
however a complete loss in enzyme activity was recorded when the enzyme
was incubated at 80 and 90Â°C for 20 and 5 min, respectively.
Nature of Buffer
Four reaction mixtures were made at pH 7.0 and received equimolar
amount of citrate-phosphate, Tris-acetate, Tris-HCl and phosphate buffers
(0.2M). Results obtained in Table 2 indicate that the activity in Tris-HCl
buffer is higher than the analogous activities obtained from the other
Effect of Freezing and Thawing
In this experiment the crude extracts of cytidine deaminase from Str.
hygroscopicus NRRL B-1476 was estimated periodically after three cycles
of freezing and thawing during a period of three days at about -5Â°C,
after which it was warmed and an aliquot was withdrawn for assay of activity
under the same experimental conditions. It is clear from Table 3 that
a slight decrease in enzyme activity about 8% after 1 cycle 24 h, on the
other hand at the end of the second and third cycles about 35 and 72%
of the activity were lost, respectively.
Dialyzing the Extracts
To find out whether or not cytidine deaminase requires a metal ion(s)
in the process of catalysis, crude enzyme extract was prepared. The extract
was dialyzed out against 200 volumes of cold 0.02 M tis-HCl buffer at
pH 7.0 for 2 h at 7Â°C. Results obtained indicate that there is an
increase of about 55% in enzyme activity upon dialysis. These results
can be interpreted by the fact that some inhibitors in the crude extract
that affect enzyme activity were dialyzed out.
||Effect of frequent freezing and thawing on cytidine deaminase activity
of Str. hygroscopicus NRRL B-1476
|Reaction mixtures contained: cytidine, 5.0 Î¼moles;
Tris-HCl buffer (pH 7.0); 100 Î¼moles; extract protein, 0.69 mg;
total volume, 1.0 mL; temperature, 50Â°C and reaction time, 60
||Effect of some activators and inhibitors on cytidine deaminase activity
of Str. hygroscopicus NRRL B-1476
|Reaction mixture contained: cytidine, 5.0 Î¼moles;
Tris-HCl buffer, pH 7.0, 100 Î¼moles; extract protein, 1.86 mg;
compound, 10 Î¼moles; total volume, 1.1 mL; temperature, 50Â°C
and reaction time, 60 min
||Evidence for the involvement of SH group in cytidine deaminase activity
Effect of Metal Ions
This experiment was carried out to find out whether or not cytidine
deaminase requires a metal ion(s) in the process of catalysis. Different
metal ion and other compounds were added to the reaction mixture containing
dialyzed extracts each at final concentration of 10-2 M. A
control reaction mixture that does not contain any of these salts was
made. Results in Table 4 showed that CuSO4, FeCl3
and HgCl2 had a complete inhibitory effect on cytidine deaminase,
while as MgSO4, BaCl2 and NaCl reduced activating
effect by 50% and the remaining metal salts showed different degrees of
inhibition depending upon the type of inhibitor and its final molarity
in the reaction mixture. Addition of CoSO4 at 10 mM to the
reaction mixture caused about 83% inhibition.
Effect of Sulfhydryl Compounds
To test whether or not SH groups are involved in the catalytic site of
cytidine deaminase of Str. hygroscopicus NRRL B-1476 different
reaction mixtures were prepared containing reduced glutathione, 2-mercaptoethanol
or iodoacetate at a final concentration of 10 mM. Results obtained are
cited as relative activities in Table 5 and indicated that the addition
of iodoacetate caused a complete inhibition. The inhibitory effect of
iodoacetate on cytidine deaminase activity can be interpreted by the fact
that iodoacetate alkylates the sulfhydryl group(s). Addition of reduced
glutathione and 2-mercaptoethanol at a final concentration of 10 mM to
the reaction mixtures had no effect on cytidine deaminase suggesting that
free sulfhydryl groups have no role in the activity of the enzyme.
||Effect of cytidine concentration on cytidine deaminase from Str.
Hygroscopicous NRRL B-1476. Reaction mixture contained (in 1 mL/vol.)
Substrate as indicated; extract protein 1.1 mg; Tris-HCl pH 7.0, 100
Î¼moles; temperature, 50Â°C and time of reaction 30 min
Determination of the Apparent Km and Ki Values
Inhibition exerted by uridine seems to be of the competitive type Fig.
5, also demonstrates classic Michaels Menten saturation kinetics.
The apparent of Km value for cytidine (under the experimental
conditions) and the apparent Ki value for uridine were calculated
from the Linweaver Burk plot and found to be 4.16 and 21.9 mM, respectively
(Fig. 5). However, the competitive inhibition of cytidine
deaminase by uridine leads to the suggestion of the existence of some
sort of control mechanisms which does not allow the conversion of all
the cytidine present into uridine and hence avoid depletion of the former
Dixan and Webb (1964).
This study represents an effort to search for the degradation of
purine and pyrimidine ribonucleosides and their bases in cell-free extracts
of some Streptomyces strains. These studies are rare in Streptomyces
strains. In order to obtain this goal, a screening study was made with
five Streptomyces cultures for their abilities to catalyze the
hydrolytic and or the deaminating activities of some purine and pyrimidine
ribonucleosides and their bases. The previous results show that no hydrolytic
cleavage of N-glycosidic bond of purine and pyrimidine ribonucleosides
in all Streptomyces strains under study, whileas the only activity
found in all Streptomyces strains was cytidine deamination, these
finding were similar to studies obtained by Elawarmy and Elzainy (1985)
in Streptomyces viridiviolaceus. On the other hand in filamentous
fungi the enzymes responsible for the degradation of ribonucleosides were
known, Ali and Elzainy (2000) reported cytidine deaminase in Aspergillus
niger NRRL3, Allam et al. (1981) reported the presence of a
pyrimidine ribonucleoside hydrolase that catalyzes of the N-glycosidic
bond of cytidine and uridine in Penicillium chrysogenum. He also
reported a broad spectrum intracellular constitutive purine and pyrimidine
ribonucleoside hydrolase in cell-free extracts of Fusarium moniliforme.
On comparing the levels of cytidine deaminase activity in the previously
mentioned five Streptomyces strains it is noted that the enzyme
was found in a relatively high amounts in extracts of Streptomyces
hygroscopicus NRRL B-1476 as compared with the other testing Streptomyces
so it was selected for further studies. Studies on the properties of cytidine
deaminase of Streptomyces hygroscopicus NRRL B-1476 showed that
optimum pH and temperature were found to be at 7.0 and 50Â°C, respectively,
these results was semi similar to results obtained by Elawamry and Elzainy
(1985) in Streptomyces viridiviolaceus that had optimal pH and
temperature at 6.5-7.5 and 70Â°C, respectively and in fungi. Bezborodov
et al. (1974) reported the presence of cytidine deaminase in P.
sizowi was optimum activity at pH values ranging from 5.5-7.0 and
optimum temperature of 75Â°C and Allam et al. (1991) reported
that the optimum pH for P. citrinum is 6.5, In addition studies
on thermal stability of cytidine deaminase of Streptomyces hygroscopicus
NRRL B-1476 indicated that enzyme completely restored its activities at
50 and 60Â°C for 30 min, however a complete loss in enzyme activity
was recorded when the enzyme was incubated at 80 and 90Â°C for 20 and
5 min, respectively. In contrast the corresponding enzyme reported in
P. politans NRC-510 extracts is unique for its thermophilic nature
as the enzyme restored its activity at 80Â°C for 60 min in absence
of substrate (Elshafei et al., 2005), in accordance with our results.
Dialyzing the extract caused an increase in enzyme activity of about 55%.
These results can be explained by the fact that some inhibitors in the
crude extracts were dialyzed out. Among the various metal salts tested,
CuSO4 and HgCl2 at a concentrations 5x10-3
M and 10-2 M had a complete inhibitory effect on enzyme activity,
while CoSO4, MnCl2 and MgCl2 caused a
remarkable activating effect. HgCl2 evidenced complete inhibition
of cytidine deaminase activity; this fact suggests the possible presence
of thiol groups at the catalytic site of the enzyme. The enzyme was affected
by sulfhydryl compounds such as reduced glutathione and 2-mercaptoethanol
which cause a slight activating effect, whileas the addition of iodoacetate
was found to be inhibitory on enzyme activity On the adverse, Allam
et al. (1991) reported that SH group(s) might not be involved in catalytic
site of that enzyme from P. citrinum. The apparent Km
and Ki values for cytidine and uridine were calculated from
the Linweaver Burk plot and found to be 4.16 and 21.9 mM, respectively.
These results similar to result obtained by Allam et al. (1991).
However, the competitive inhibition of cytidine deaminase by uridine leads
to the suggestion of the existence of some sort of control mechanisms
which does not allow the conversion of all the cytidine present into uridine
and hence avoid depletion of the former.
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