(Received for publication, March 29, 1995; and in revised form, May 1, 1995)
From the
The oleandomycin (OM) producer, Streptomyces antibioticus, possesses a mechanism involving two enzymes for the intracellular
inactivation and extracellular reactivation of the antibiotic.
Inactivation takes place by transfer of a glucose molecule from a donor
(UDP-glucose) to OM, a process catalyzed by an intracellular
glucosyltransferase. Glucosyltransferase activity is detectable in
cell-free extracts concurrent with biosynthesis of OM. The enzyme has
been purified 1,097-fold as a monomer, with a molecular mass of 57.1
kDa by a four-step procedure using three chromatographic columns. The
reaction operates via a compulsory-order mechanism. This has been shown
by steady-state kinetic studies using either OM or an alternative
substrate (rosaramycin) and dead-end inhibitors, and isotopic exchange
reactions at equilibrium. OM binds first to the enzyme, followed by
UDP-glucose. A ternary complex is thus formed prior to transfer of
glucose. UDP is then released, followed by the glycosylated
oleandomycin (GS-OM).
The biosynthesis of a potentially lethal antibiotic necessitates
the existence of a self-resistance mechanism in the producing organism.
One of the most common (for review, see (1) ) involves
modification of the antibiotic target site, for example, inhibitors of
ribosomal
function(2, 3, 4, 5, 6, 7) ,
RNA polymerase(8, 9, 10) , DNA gyrase (11) , elongation factor EF-Tu(12) , and fatty acyl
synthase(13) . Activities capable of inactivating antibiotics in vitro have also been described. However, despite the
structural diversity of the different antibiotic families, most of the
inactivating activities so far described catalyze the N-acetylation of amino groups or O-phosphorylation of
hydroxyl groups using acetyl-coenzyme A and ATP, respectively, as donor
cofactors. Most of these activities have been reported in
aminoglycoside producers (reviewed in (1) ). Inactivation of
fosfomycin by a glutathione S-transferase in the producer
strain has also been reported(14) . A possible role for the
inactivating activities in the producer strains is their participation
in the antibiotic biosynthetic pathway, being in fact biosynthetic
enzymes. In the case of puromycin, biochemical evidence suggests a role
for puromycin N-acetyltransferase in the biosynthesis of the
antibiotic(15) . We have previously reported the existence
of a dual mechanism for inactivation and reactivation of OM in the
oleandomycin (OM) (
Figure 1:
Chemical structure of OM and proposed
site of action (indicated by the arrow) for the
GTF.
Very little is known at
present about the reaction mechanisms of enzymes involved in antibiotic
biosynthesis and resistance. Here we report the purification and
characterization of the OM GTF and kinetic studies to obtain a more
detailed knowledge of the enzymatic mechanism of this GTF.
Figure 2:
Time
course for the biosynthesis of OM (
Figure 3:
Chromatography of the different steps in
the purification of the GTF. Elution profile from (A) the
Q-Sepharose column, (B) the Sephacryl S-200 column, and (C) the UDP-glucuronic acid agarose column.
Figure 4:
Silver-stained SDS-PAGE analysis of the
different steps in the purification of the GTF activity. Lane
A, cell-free extract; lane B, ammonium sulfate
precipitation; lane C, active fractions eluted from the
Q-Sepharose column; lane D, active fractions eluted from the
Sephacryl S-200 column; lane E, active fractions eluted from
the UDP-glucuronic acid agarose.
where A and B signify the first and second
substrates, respectively, that bind to the enzyme, V is V
Figure 5:
Double-reciprocal plots of the initial
velocities with variable substrate concentrations. A, variable
UDP-Glc at fixed OM concentrations.
Several assays were performed in
which one of the substrates (OM) was replaced by rosaramycin.
Rosaramycin is a macrolide that is related structurally to OM and has
been described as an alternative substrate for the GTF(16) .
When rosaramycin was used instead of OM, convergence between lines
became more apparent (data not shown), due to the change in the kinetic
parameters. The values of the new parameters obtained by nonlinear
regression were: V = 232 ± 31
nmol However, a graphical analysis of the results of both experiments
expressed in reciprocal form in terms of Ø's according to
the equation of
Daziel(25) :
showed that all the values of the Ø's changed when
rosaramycin was used as alternative substrate. This indicates OM binds
the enzyme in the first place (substrate A), based on the fact
that the use of an alternative substrate for B would maintain
some of the coefficients constant depending on the mechanism of the
reaction. It can also be concluded that the reaction occurs by a
compulsory-order mechanism, due to the fact that in the random order
equilibrium pathway Ø
The antibiotic erythromycin is structurally very
similar to OM, but it is not a substrate for the GTF(16) . When
erythromycin was added to the reaction using OM as the varied
substrate, a set of lines of different slopes was observed, while the
intersection point remained constant, thus indicating a competitive
inhibition (Fig. 6A). If the substrate varied was
UDP-Glc, the lines changed, both in their slope and intersection point (Fig. 6B), implying noncompetitive inhibition.
Figure 6:
Patterns of dead-end inhibition of the GTF
by erythromycin (panels A and B) or by UTP (panels C and D) as a function of the OM (panels
A and C) or UDP-Glc (panels B and D)
concentrations. The concentrations of UDP-Glc and OM were kept constant
at 8 µM (panels A and C) and 2
µM (panels B and D), respectively. The
concentrations of erythromycin as inhibitor (panels A and B) were:
UTP
was also found to act as a dead-end inhibitor of the reaction. When
reactions were carried out in the presence of UTP using UDP-Glc as the
varied substrate, the lines changed their slopes but the intersection
point remained constant, suggesting competitive inhibition (Fig. 6D). If the varied substrate was OM, the lines
were parallel, with unchanged slope but with changed point of
intersection, indicating uncompetitive inhibition (Fig. 6C). Data from dead-end inhibitors are
summarized in Table 2. The patterns of inhibition observed showed
noncompetitive inhibition for erythromycin when UDP-Glc is varied and
uncompetitive inhibition for UTP when OM is varied, and this is
compatible only with a compulsory-order mechanism. It does not,
however, differentiate between formation of a ternary complex or if its
concentration is negligible (Theorell-Chance mechanism). On the other
hand, these results allow confirmation of the order of substrate
addition, OM being first and UDP-Glc second.
Figure 7:
Plot of
When the
effect of increased concentrations of the pair OM/GS-OM on the rates of
exchange between UDP-[
Figure 8:
Influence of increasing concentrations of
different substrate/product pairs on the rate of exchange between
UDP-Glc and GS-Om. A:
Figure S1:
Scheme 1.
The differentiation between a
compulsory-order system with the formation of a ternary complex and a
Theorell-Chance mechanism is possible by performing experiments where
the influence on the rate of isotopic exchange is measured when the
concentration of the pairs composed by the second substrate and one of
the different products is increased. Increasing the concentration of
the UDP-Glc/GS-OM pair a hyperbolic plot was produced (Fig. 8B), while the increase of the UDP-Glc/UDP pair
produced a plot which initially increased but later showed a depression
at higher concentrations (Fig. 8B). These results
confirm the order of the product release from the enzyme, since raising
the concentrations of the UDP-Glc/UDP pair increases the concentrations
of the central enzymic forms, and depresses the rate of isotopic
exchange. This is compatible only with an ordered mechanism in which a
ternary complex is formed. In a Theorell-Chance mechanism, the increase
in the UDP-Glc/UDP pair would produce a hyperbolic plot since in this
mechanism the process EA + B EQ &lrhar2; + P is a simple
bimolecular process. In summary, the mechanism of the GTF reaction
can be represented as in Fig. S1. The substrates bind in a
compulsory order to the enzyme, first the OM and then the UDP-Glc to
form a ternary complex in which the exchange reaction takes place.
Product release also occurs in a compulsory order, UDP first followed
subsequently by GS-OM.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)producer, Streptomyces
antibioticus(16) . This strain synthesizes an
intracellular glucosyltransferase (GTF) enzyme capable of inactivating
OM (and a few other macrolides) by glucosylation of the 2`-hydroxyl
group of the desosamine present in the antibiotic (Fig. 1). This
glucosylated oleandomycin (GS-OM) is the final intracellular product of
the pathway. We have proposed that this inactive GS-OM could be
secreted through participation of ABC (``ATP Binding Cassette'') transporters encoded by the oleB and oleC genes(18) . In agreement with this, recent
experimental evidence has demonstrated that the OleB protein is capable
of pumping out GS-OM(17) . To complete OM biosynthesis, S.antibioticus synthesizes and secretes a second enzyme
that has recently been purified(19) . This reactivates the
antibiotic by catalyzing release of glucose.
Reagents
Oleandomycin, erythromycin, and
UDP-glucuronic acid agarose were purchased from Sigma. Rosaramycin was
purchased from Schering Corp. UDP-[6-H]glucose
(specific activity 7.6 Ci/mmol, 1 mCi/ml) from Amersham International
(United Kingdom). UDP-glucose, UTP, and dithiothreitol (DTT) were from
Sigma. Acrylamide and bisacrylamide were from Bio-Rad. Acetonitrile
(HPLC grade) and ammonium sulfate from Merck, and Q-Sepharose,
Sephacryl S-200, and Superdex 75 from Pharmacia Biotech Inc. (Uppsala,
Sweden). All other chemicals were obtained from commercial sources and
were of analytical grade.
Purification Procedure
S. antibioticus ATCC 11891, an OM producer, was grown in 2-liter
Erlenmeyer flasks containing 500 ml of TSB (tripticasein soy broth,
Oxoid) liquid medium. The cultures were inoculated with 50 µl of a
dense spore suspension and after 48 h at 30 °C on an orbital shaker
incubator (200 rpm), the mycelia were collected by filtration through
Whatman No. 1 filters. The mycelial paste was washed twice with buffer
A (50 mM Tris-HCl buffer, pH 8.0, 1 mM EDTA, and 1
mM DTT). The mycelia was then disrupted by homogenization with
glass beads (0.10 mm in diameter) in a Bead Beater (Bead Beater,
Bartlesville, OK) for 10 15-s periods with 3-min intervals for
cooling. Unbroken cells and debris were removed by centrifugation at
14,000
g for 30 min. Nucleic acids were precipitated
with streptomycin sulfate (1% final concentration) and the supernatant
fractionated by precipitation with ammonium sulfate at 50% saturation.
After centrifugation (14,000
g, 30 min), the
supernatant was dialyzed against buffer A and applied to a Q-Sepharose
column (200 ml volume) at a flow rate of 3 ml/min. The column was
eluted with a gradient of 0-1 M NaCl. Active fractions
were concentrated by ammonium sulfate precipitation (95% saturation)
and, after centrifugation, the precipitate was resuspended in 5 ml of
buffer B (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, and 0.5 M ammonium sulfate) before dialysis against
buffer B. The sample was then applied to a Sephacryl S-200 column (2.6
90 cm) at a flow rate of 0.3 ml/min. Active fractions were
dialyzed against buffer C (25 mM Tris-HCl, pH 7.5, 1
mM DTT, and 20% glycerol) and then applied to a UDP-glucuronic
acid-agarose column (10 ml) at a flow rate of 0.3 ml/min. Elution was
with a 0-1 M NaCl gradient in buffer C.
Enzyme Assays
OM inactivation was monitored using
a reaction mixture containing (for a 50-µl final volume): 1 µl
of OM (3 µM final concentration), 1 µl of
UDP-[H]Glc (0.26 µM final
concentration), and 1 µl of UDP-Glc (22 µM final
concentration), and a variable volume of each fraction or buffer. The
mixture was incubated at 30 °C for 3 min and the pH subsequently
raised by addition of 5 µl of 0.2 M NaOH. It was then
extracted with 50 µl of chloroform, following centrifugation at
12,000
g for 5 min in an Eppendorf minifuge, 25 µl
of the lower organic phase were evaporated to dryness and resuspended
in 50 µl of methanol. Any radioactivity measured was indicative of
the inactivating activity.
Biological Assay of OM
The biological activity of
OM was tested by performing a bioassay against Micrococcus luteus ATCC 10240(20) .Purification of GS-OM
After performing
glucosylation reactions as described above, the pH of the reaction
mixture was raised by adding 0.2 M NaOH (0.1, v/v). This was
extracted with 1 volume of chloroform. After centrifugation, the
organic phase was recovered, flash evaporated, and suspended in
methanol:water (1:1 by volume), before application to a µBondapak
C column. GS-OM and OM were separated using an isocratic
gradient composed of 30% acetonitrile and 70% 50 mM phosphate
buffer, pH 6.8, at a 2 ml/min flow rate. Detection was followed by
measuring absorbance at 200 nm. Fractions corresponding to GS-OM were
collected, dried, and resuspended in chloroform so as to eliminate
residual phosphate. The chlorofom was then removed by evaporation and
the residue (containing the GS-OM) dissolved in methanol:water (1:1 by
volume) and stored at -20 °C prior to use.
Polyacrylamide Gel Electrophoresis and Protein
Analysis
Analysis of the samples during protein purification was
performed using SDS-PAGE(21) . Protein estimation was carried
out by measurement of absorbance at 280 nm and by a protein-dye binding
assay (22) .Steady State Kinetics
Initial velocity assays were
carried out using the radiolabeled assay described above. The
concentration of one substrate was varied while the concentration of
the second substrate was held at a nonsaturating concentration. After
stopping the reaction, the amount of GS-OM produced was estimated. The
experimental data reported here were all carried out at least in
duplicate. Data were analyzed by linear and nonlinear regression using
the program Statistica for Windows (Statsoft, Inc.).Measure of Isotopic Exchange Reactions Catalyzed by the
GTF
The exchange reactions between
UDP-[H]Glc and [
H]GS-OM
were assayed by measuring the formation of
[
H]GS-OM from UDP-[
H]Glc
and unlabeled OM. Assays were performed as described under
``Steady State Kinetics,'' except that the concentrations of
unlabeled reactants and products were adjusted to the equilibrium
conditions at the beginning of the experiment. The concentrations for
the unchanged pair were 0.64 and 5 µM, and the
concentrations of the changed pair were varied from 0.5 to 40 times the
concentration of the other pair depending on the assay. After addition
of enzyme, the reaction mixture was incubated at 30 °C for 15 min.
A small concentration (10 nM) of
UDP-[
H]Glc was added subsequently, and samples
taken at different times and used to calculate the rate of isotopic
exchange.
Time Course for the Synthesis of OM and the
GTF
OM production began 24 h after inoculation of S.
antibioticus in TSB medium (Fig. 2) and increased
progressively during the following 24 h. In cell-free extracts GTF
activity was also detected after 24 h of growth, which coincided with
the beginning of OM biosynthesis. The GTF activity increased during the
next 24 h following a similar pattern to that of OM biosynthesis,
suggesting, therefore, that both events are coordinately regulated.
) and the OM GTF
([circo]) during growth of S.antibioticus. At different times of incubation of S.antibioticus in TSB medium, samples were removed and OM present in the
supernatant determined by bioassay against M.luteus.
The mycelia was collected and the GTF activity assayed using a
cell-free extract (see ``Materials and
Methods'').
Cellular Location of the GTF
To determine the
physical location of the GTF, protoplasts were prepared by lysozyme
digestion (23) and gently lysed by 1:10 dilution into a
nonstabilizing buffer (50 mM Tris-HCl, pH 8.5, 1 mM
DTT) containing either 150 mM or 1 M NaCl. The
samples were ultracentrifuged (100,000 g) for 30 min
and the GTF activity assayed in fractions along the tube. Activity was
distributed uniformly, and was independent of the salt concentration
used in protoplast lysis (data not shown), indicating the absence of
any association between GTF and particulate fractions (i.e. membranes).
Purification and Characterization of the GTF
The
GTF enzyme was purified by a four-step purification procedure using
three chromatographic columns (Fig. 3). A summary of the
purification procedure is shown in Table 1. The enzyme was eluted
from the Q-Sepharose column at 0.33 M NaCl (Fig. 3A) and from the UDP-glucuronic acid-agarose
column at 0.12 M NaCl (Fig. 3C). The enzyme
was purified 1,097-fold with an 18% yield. It showed a molecular mass
of approximately 57.1 kDa as deduced from SDS-PAGE gels (Fig. 4)
and 56 kDa as calculated by gel filtration on a Superdex 75 column in
the presence of 0.15 M NaCl at a flow rate of 0.1 ml/min (data
not shown). These experiments suggests that the GTF is a monomer. The
enzyme was moderately unstable when kept at low temperature, losing
almost all activity after a few days at 4 °C, although the addition
of 1 M ammonium sulfate or 20% glycerol was found to stabilize
the enzyme. Activity was maximal between pH 8 and 8.5. Enzyme activity
was shown to be unaffected by the addition of different mono or
divalent cations, but was affected by the addition of low
concentrations of several organic solvents such as methanol, ethanol,
butanol, and acetone. This contrasts with the purified extracellular
glycosidase which is active in the presence of as much as 20% acetone
or ethanol(19) .
, GTF
activity;
, salt gradient; --, absorbance
at 280 nm.
Steady State Initial Velocity Studies
For most of
the reaction mechanisms involving a two-substrate enzyme, the initial
rate is given, according to the nomenclature of Cleland(24) ,
by the following equation:
, K
and K
are the K
values for A and B, respectively, and K
is the dissociation constant for the
reaction of A with the free enzyme. A plot of 1/vversus 1/[substrate] showed a set of converging
lines, when the substrate was either UDP-Glc (Fig. 5A)
or [OM] (Fig. 5B) at fixed concentrations of
the other substrate. The following parameters were obtained by
performing nonlinear regression analysis of the initial velocity data: V = 363.8 ± 27
nmol
min
mg protein
; K
= 2.9 ± 0.19
µM, K
= 21.57
± 2.3 µM, K
=
0.44 ± 0.06 µM. A set of intersecting lines in the
analysis of the initial velocity steady state kinetic data is
indicative of a sequential mechanism in which a ternary complex is
formed prior to product release.
, 7 µM;
,
3.5 µM; ▪, 2.3 µM;
, 1.75
µM; ▴, 1 µM. B, variable OM at
fixed UDP-Glc concentrations.
, 32 µM;
, 16
µM; ▪, 8 µM;
, 5.2
µM; ▴, 3.1 µM;
, 2.2
µM.
min
mg protein
, K
= 13.22 ± 2.1
µM, K
= 42.09
± 3.8 µM, K
=
11.14 ± 1.6 µM. An increase in the value of K
close to that of K
makes the convergence between the lines much more evident
and places the intersection point just below the horizontal axis.
/Ø
might be
unaltered when using an alternative substrate for A.
Study of Substrate Binding Order Using Dead-end
Inhibitors
Dead-end inhibitors were used to obtain information
about the mechanism of the reaction and the substrate binding order.
The concentration of one of the substrates was fixed at a subsaturating
level, and the concentration of the other substrate varied; experiments
were performed at different concentrations of the dead-end inhibitor,
including 0.
, 0 µM;
, 2.5
µM; ▪, 5 µM;
, 10
µM. The concentrations of UTP as inhibitor (panels C and D) were:
, 0 µM;
, 25
µM; ▪, 67 µM;
, 200
µM.
Equilibrium Constant
The equilibrium constant for
the GTF reaction might be expected to be independent of pH as no net
production or uptake of protons occurs during the course of the
transglucosylation reaction. The time taken to reach equilibrium was
first established by measurement of the change in the concentration of
GS-OM with time after addition of the enzyme to solutions similar to
those described below. Reaction was always completed in less than 1 h.
When sampled after several hours, the concentration of GS-OM began to
decrease, due to its instability at 30 °C. K was measured by fixing the ratio of
[OM]/[GS-OM] at 1 and varying the ratio of
[UDP]/[UDP-Glc] from 10 to 80. A plot of the change
in GS-OM concentration versus [UDP]/[UDP-Glc] is shown in Fig. 7. The
value at which the line intercepts with the ordinate axis for an
abscissa value of 0 is equal to the equilibrium constant K
= 60.81.
[GS-OM] versus [UDP]/[UDP-Glc] for determining the
equilibrium constant of the GTF reaction. A linear regression of the
data gave a [UDP]/[UDP-Glc] value of 60.81 at a
[GS-OM] of 0.
Reactions of Isotopic Exchange at Equilibrium
To
obtain information about the order of products release and more details
about the reaction mechanism, we performed isotopic exchange
experiments under equilibrium conditions. The concentration of a
substrate-product pair was varied so as not to disturb the equilibrium;
its effect on the rate of isotopic exchange between
UDP-[H]Glc and [
H]GS-OM was
then determined. The progress curve for the exchange, monitored by
sampling the reaction mixture at different time intervals, showed in
all the cases a first-order rate law; these curves were used to
calculate the rates of exchange. The exchange rate was also directly
proportional to enzyme concentration, as would be expected in the
absence of subunit dissociation and association effects.
H]Glc and
[
H]GS-OM was measured, a hyperbolic plot was
obtained (Fig. 8A). A similar experiment varying the
pair OM/UDP produced a plot showing an initial increase followed by a
decrease due to inhibition of the isotopic exchange at high
concentrations of the pair OM/UDP (Fig. 8A). These
results are consistent only if the order of product release is the one
shown in Fig. S1, where UDP leaves first followed by GS-OM.
Raising the concentration of the pair OM/UDP makes the enzyme-GS-OM and
enzyme species scarce, increasing other forms, and the exchange rates
are lowered. If the concentration of the OM/GS-OM pair was raised, only
the free enzyme becomes scarce and the rates of exchange approach a
maximum asymptotically.
, varying the pair OM/UDP;
,
varying the pair OM/GS-OM. B:
, varying the pair
UDP-Glc/UDP;
, varying the pair
UDP-Glc/GS-OM.
We thank Schering Corp. for the kind gift of
rosaramycin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.