Controlling selection pres-sures on the enzyme, by controlling the environment of the host organism during the evolution of resistance [4], allows control over the number of mutations in
Trang 1in Escherichia coli
Morgan Watson, Jian-Wei Liu and David Ollis
Research School of Chemistry, Australian National University, Canberra, Australia
Naturally occurring enzyme inhibitors are found in
every living organism, serving purposes ranging from
the regulation of metabolism to weapons against
com-petitors, predators and prey By contrast, artificial
enzyme inhibitors serve two primary purposes; to aid
the study of enzymes and biological pathways, and to
serve as drugs in medical applications [1] The synergy
between these two approaches is well illustrated when
examination of the means by which an enzyme becomes
resistant to an antagonist furthers our understanding of
the functionality of the enzyme itself However, the
study of the mechanistic relationship between inhibition
by antibacterials and enzymatic activity is complicated
by the systematic differences between susceptible and
resistant forms of an enzyme Clinical isolates typically
have multiple mutations present [2] and in some cases
bear little resemblance to the native form [3] so it is
dif-ficult to determine the effect of any one mutation
Although we can examine the result of the mutations,
we can only postulate about the reasons that gave rise
to them Directed evolution provides a mechanism for
simplifying these studies Controlling selection pres-sures on the enzyme, by controlling the environment of the host organism during the evolution of resistance [4], allows control over the number of mutations intro-duced, and ensures that the resistant forms produced are related to the susceptible native form This allows
us to determine additional information on why the mutations arose and in what order multiple mutations accumulated, information that is not always available for samples isolated from other sources Screening tech-niques used in directed evolution also enable us to pro-duce multiple mutant forms of the enzyme, all of which have potentially used different methods to overcome the obstacles present in the directed evolution process This allows for a greater understanding of enzyme mechanisms than studies of clinical isolates Directed evolution is gaining popularity as a means of studying enzymes and has recently been used to examine other antibacterial resistance systems [5]
Because of its central role in one-carbon metabo-lism, dihydrofolate reductase (DHFR) has long been a
Keywords
antibiotic resistance; dihydrofolate
reductase; directed evolution; trimethoprim
Correspondence
M Watson, Research School of Chemistry,
Australian National University, Canberra,
ACT 0200, Australia
Fax: +61 261 250 750
Tel: +61 261 258 017
E-mail: watson@rsc.anu.edu.au
(Received 2 January 2007, revised 12 March
2007, accepted 22 March 2007)
doi:10.1111/j.1742-4658.2007.05801.x
Directed evolution is a useful tool in the study of enzymes It is used in this study to investigate the means by which resistance to the antibiotic trimeth-oprim develops in dihyrofolate reductase from Escherichia coli Mutants with clinical levels of resistance were obtained after only three generations After four generations of directed evolution, several mutants were charac-terized, along with some point mutants made to investigate amino acid changes of interest Several mutations were found to grant resistance to trimethoprim, both by reducing the binding affinity of the enzyme for the drug, and by increasing the activity of the enzyme
Abbreviations
DHF, 5,6-dihydrofolate; DHFR, dihydrofolate reductase; MHA, Mueller Hinton agar; MIC, minimum inhibitory concentration; THF,
5,6,7,8-tetrahydrofolate; TMP, trimethoprim.
Trang 2target for drugs [6,7] The structure and function of
native forms of DHFR from a variety of sources have
been studied DHFR from Escherichia coli is a
mono-meric protein of 18 kDa The structure of the native
form has been determined, including complexes with
both substrates and a number of inhibitors [8] DHFR
catalyses the reduction of NADPH and
5,6-dihydrofo-late (DHF) to NADP+and 5,6,7,8-tetrahydrofolate
(THF), via the redox reaction:
DHFþ NADPH þ Hþ! THF þ NADPþ
Binding of the substrates is ordered, with the reaction
proceeding by the binding of first NADPH then DHF,
followed by the release of NADP+and binding of
another NADPH molecule before the release of THF
THF release is the rate-limiting step in the reaction [9]
DHFR is also capable of reducing folate to DHF,
although at a considerably slower rate than the
reduc-tion of DHF to THF This reducreduc-tion is essential for
maintaining the cellular pool of reduced folate
required to synthesize thymidylate, purines and
methi-onine [10,11] Compounds used to inhibit DHFR are
collectively know as antifolates, and are used to treat a
variety of conditions ranging from bacterial infections
and malaria to cancer This study uses the antifolate
drug trimethoprim (TMP) (Fig 1) TMP is used as an
antibiotic as it has a high degree of specificity for
bac-terial DHFRs over eukaryotic DHFRs
The widespread use of antifolates has lead to the
development of resistance to many of the compounds
[12,13], and numerous studies of DHFR from many
organisms [11] The breadth of information available
on DHFR and the significance of it in the treatment
of a range of conditions makes it an ideal subject for examining the ability of directed evolution to aid us in understanding and overcoming the rise of resistance to enzyme inhibitors
In this study, we used directed evolution to generate
a library of E coli DHFR mutants with resistance to TMP Selected mutants were characterized kinetically using fluorescence techniques Third- and fourth-gen-eration mutants were found which possessed greatly increased levels of TMP resistance, with only minor deleterious effects on activity, and in many cases improvement of either substrate Kmvalues or kcat The location and probable effect of repeatedly occurring mutations has been examined
Results and Discussion
Library generation Each round of evolution libraries consisted of
100 000 cfu Of these, 250–300 colonies were selected for secondary screening, and the best 20–25 colonies selected for sequencing From those se-quenced, 10–15 mutants were shuffled to produce the next round of mutants Amino acid changes observed in the sequenced mutants are shown in Table 1 There were a few false positives so the numbering of mutants is not necessarily sequential
No duplicates were seen, presumably because of the large number of potential combinations of generated mutants
Fig 1 Comparison of the structure of 5,6-dihydrofolate (DHF) and trimethoprim (TMP) Of particular interest is the substitution of an amine group on TMP for the double bonded oxygen in DHF (circled).
Trang 3Table 1 List of mutants generated Mutations found in those resistant colonies sequenced Only mutations that occur more than once are shown, this may result in some mutants appearing identical The percentage of mutants containing a given mutation in each round is indicated.
(L ⁄ Q)
(W ⁄ Q)
(L ⁄ Q)
(W ⁄ Q)
(L ⁄ Q)
(W ⁄ Q)
Trang 4The genes sequenced at the end of the first
genera-tion contained only one or two mutagenera-tions each
With the exception of F53S, these mutations
recom-bine in subsequent rounds so that at the end of four
rounds a number of these mutations (M20V, A26T,
I94L) occurs in most of the resistant enzymes The
A26T and H45R mutations only become common in
the third and fourth generations One of these,
H45R, originated only in the third round, but
quickly became common The favouring of these
mutations at higher levels of TMP provides a good
indication that they either confer a high level of
resistance, or are necessary to allow other mutations
that confer such resistance
There are three mutations that vanish from later
rounds after being common in earlier ones; P21L,
P21Q, and W30R It is possible that these are too
inef-fective to survive in later rounds, or it may be that
although these mutations are capable of protecting the
bacteria from TMP, they are incompatible with the
M20V mutation (never occurring together in Table 1)
and are lost as the M20V mutation (shown to be a
considerably more effective mutation on Table 2)
becomes more common Other mutations show neither
affinity nor conflict with each other Not shown in
Table 1 are mutations that only occurred in one
mutant, demonstrating little selective advantage
Protein expression and purification
All mutants were expressed to between 80 and 100%
of the level of native DHFR, so we can conclude that
the observed resistance during selection was not due to
increased overexpression All enzymes were purified to
> 95% pure, as judged by visual inspection of a Coo-massie Brilliant Blue-stained SDS gel
Kinetic assays Kinetic constants determined from the initial rate reactions are shown in Table 2 These were deter-mined using Michaelis–Menten plots of the data collected Km values obtained for the native form are consistent with those available in the literature (0.7–3.2 lm for DHF and 0.94–6.8 lm for NADPH)
as were the kcat values (literature values of 18–29Æs)1) [10,14–16]
With two exceptions, the kinetic constants measured for DHF and NADPH binding of the mutants show a remarkable similarity to that of the native form This suggests that the native activity is the minimum required for viable cells Two mutants showing an improvement on these constants, 3-20 and 4-4, both had significantly increased Km values for NADPH Both of these, along with several other mutants, had
an increased kcat This may, to some extent, compen-sate for the increased Km values The prevalence of increased kcat values in both directed evolution and single-site mutants suggests that it may also play a direct role in TMP resistance Many of the single-site mutants possess kcat values far higher than those of the directed evolution mutants that contain them This
is especially true for the third generation, whereas mutants in generation four have more comparable kcat
values This leads to the conclusion that the effects of the single-site mutants are not additive, and are cap-able of interfering with each other Only the fourth-round generation selection conditions were sufficiently
Table 1 Continued.
(L ⁄ Q)
(W ⁄ Q)
(L ⁄ Q)
(W ⁄ Q)
Trang 5stringent to select combinations of mutations
compar-able to the single-site mutants
Binding assays
Binding constants are given in Table 2 A significant
increase in Kd is apparent between generations three
and four However, the Kd of 3-20 is only a little
higher than that of the native form, begging the
ques-tion of why this mutant was selected and how it has
achieved a minimum inhibitory concentration (MIC)
value comparable with other TMP-resistant mutants
The reason for this would appear to lie in the kcat
value for 3-20, the highest for all round three mutants
In this case, it would appear that an increased kcat
value is capable of conferring resistance to the effects
of TMP, even in the presence of a Kdthat is not
signi-ficantly different from the native DHFR
The TMP binding constants for the single-site
mutants reveals some interesting results The very high
Kdvalues for M20V and H45R suggest that a far more
stringent set of selection methods could be applied
ear-lier, and the comparatively low Kdvalues for the
direc-ted mutants is a result of insufficient evolutionary
selection More interesting is that these Kd values can
be obtained with little loss of substrate binding and
kcat Combination of M20V and H45R with others
appears to result in the lowering of Kd, compensated
for by increases in kcat, allowing the enzyme to exploit both paths to resistance
Stability assays All enzymes tested had a t50 (defined as the tempera-ture required for irreversible loss of 50% of maximal activity after 30 min incubation) of 56 ± 4C There was no significant difference between the wild-type
E coli DHFR and any of the mutants (Table 2) Sta-bility was monitored for its potential to explain anti-biotic resistance in terms of protein stability rather than enhanced kinetics However, it appears that native E coli DHFR is already a relatively stable enzyme and the mutations examined in this study had little effect on this stability The temperatures required
to cause an irreversible loss of activity in the enzyme are clearly higher than those experienced by the enzyme at any point in this study or in the cell Main-taining this level of stability may be a requirement to produce viable cells
MIC tests Susceptibility tests revealed an increased resistance to TMP for all mutants, and although the degree of over-expression resulting from our choice of vector makes direct comparisons with the MIC values for TMP
Table 2 Kinetic constants of analysed mutants Mutations present in the directed evolution mutants are listed Errors indicated are standard errors MIC and t 50 determinations were only preformed on directed evolution mutants Methods of determination of kinetic values are given
in the Experimental procedures Values given are the means of three determinations, and the errors are the standard deviation of the results.
Mutant
Km
(DHF)
l M Error
Km (NADPH)
k cat
s)1 Error
Kd (TMP)
n M Error
MIC lgÆmL)1
t 50
C Error Mutations
3-2 0.890 0.025 1.460 0.063 29.06 3.28 39.5 4.5 100 57.6 1.3 M20V, A26T, H45R,
A84V, I94L, I115V 3-3 0.885 0.039 1.480 0.072 29.23 6.10 108.4 22.6 > 200 55.0 0.9 M20V, A26T, I115V, F153S 3-6 0.332 0.017 0.592 0.022 13.27 0.05 54.8 0.2 > 200 56.5 1.0 A26T, W30R, I94L, K109R 3-20 2.230 1.750 6.100 2.720 289.90 106.79 13.3 4.9 100 57.9 1.5 W30R, I94L
4-1 0.998 0.045 0.156 0.027 22.29 2.66 876.5 104.7 100 57.7 0.8 M20V, A26T, H45R, I94L,
K109R, P129L, A144T, R158W 4-4 2.110 0.612 3.100 0.819 246.91 42.50 242.4 41.7 200 55.8 1.1 M20V, A26T, I94L, R159G 4-9 2.220 0.209 0.987 0.104 201.93 3.04 938.6 14.1 > 200 57.7 0.6 S3R, V10A, M20V, A26T, H45R,
Q65R, I94L, K109R, E154K 4-18 1.560 0.126 0.880 0.064 431.61 35.39 776.1 63.6 200 55.8 1.1 M20V, A26T, W74C, I94L, D116Y V10A 0.494 0.021 1.860 0.033 368.81 89.73 332.8 81.0
M20V 1.180 0.077 0.523 0.022 65.04 1.04 1096.7 17.6
A26T 0.327 0.036 0.449 0.023 120.23 8.08 166.2 11.2
W30R 0.272 0.120 2.020 0.203 264.10 0.31 132.5 0.2
H45R 0.501 0.054 0.070 0.053 150.39 3.37 618.4 13.8
I94L 2.060 0.707 1.230 0.672 438.60 14.38 160.9 5.3
Trang 6reported for clinical isolates difficult, comparisons are
still possible Other studies have shown susceptible
forms of DHFR to be inhibited by TMP
concentra-tions in the 1–2 lgÆmL)1range [17], the same as
deter-mined in this study for native protein in our
overexpression system It is also worth noting that
TMP-resistant isolates from other studies were
resist-ant to concentrations of TMP in the range of 16–32
times that of the susceptible form [17] Some of the
DHFR mutants generated in this study were resistant
to TMP concentrations of more than 100 times that of
the native enzyme, so that it is very likely that these
would be of clinical significance
Although one would intuitively expect these
increa-ses to parallel those of the mutant Ki values, this is
not the case Cellular resistance appears to be
depend-ant on all the kinetic properties of the enzyme, not
just its affinity for TMP This is illustrated with the
increase in kcatin many mutants and single-site
muta-tions, especially in the case of 3-20, where TMP
resist-ance appears solely dependant on the increase in kcat
noted above
Single-site mutations: structure⁄ function
correlations
Many of the point mutants examined are located
in close proximity to either the active site, or in the
FG and M20 Loops (Fig 2) As the role of these loops
in catalysis of the reduction of DHF has been well
established [8], the manner in which these residues can
impact the binding and activity of the enzyme are
rel-atively straightforward The role of more distantly
located residues is less easily discerned No region of
the active site remains untouched by the mutations,
with the examined point mutations spread across the
binding residues for both DHF and NADPH, as well
as affecting the M20 loop
Of the single-site mutants examined in this study, four have been identified previously as mutation sites
in clinically isolated TMP-resistant genes These four are V10A, M20V, W30R and I94L [2,18] Two of these, M20V and I94L, become very common in later generations of this evolution, while V10A maintains a steady presence and W30R starts out strong, but is eventually ousted by M20V, which it appears to be incompatible with
Val10Ala Although located distant to the active site, potential exists for this mutation to have an effect on the binding of substrates due to the movement of the M20 and FG loops during catalysis [8] Alteration of the conformation of these loops at any stage in the catalytic cycle could account for the observed differ-ences in kinetics
Met20Val This mutation falls on the highly studied M20 loop mentioned above, and is known to play an import-ant role in the catalytic cycle of DHFR [8] Although this mutation gives the greatest improve-ment in TMP resistance of all the point mutants studied, it is also the only point mutant to have a lower kcat than the wild-type enzyme This pair of effects match well with the frequency data shown in Table 1, as the concentration of TMP used in the selection process increases, so too does the frequency
of M20V, as the trade off between resistance and activity shifts to favour resistance
Fig 2 Line representation of E coli DHFR.
b sheets are shown as arrows, a helices are shown as rectangles Locations of those mutations listed in Table 1 are indicated as grey discs The binding sites for NADPH and DHF ⁄ TMP are indicated.
Trang 7Although located near the M20 loop, this mutation
has a markedly different effect to M20V Although
it confers an unremarkable level of resistance, it
improves the binding of both DHF and NADPH
The kcat of the A26T point mutant is also relatively
high compared with other point mutants It is likely
that the primary role of this mutation is not to
pro-vide resistance through reduced affinity for TMP,
but to help negate some of the negative effects other
mutations have on the activity of DHFR
Trp30Arg
The substitution of arginine for tryptophan at
resi-due 30 introduces an additional -NH3- moiety into
the DHF-binding site (while maintaining the existing
-NH2- group already present), in proximity to a
dou-ble-bonded oxygen residue possessed by DHF but
lacking in TMP (as shown in Fig 1) This allows
for the formation of an additional hydrogen bond
with DHF, stabilizing its binding, while destabilizing
the binding of TMP The net result of this is a
kin-etic profile similar to that of A26T Unlike A26T,
W30R would appear to be incompatible with M20V,
never occurring in the same mutant, and eventually
being lost as the frequency of M20V increased The
reason for this incompatibility appears to lie in the
binding of DHF⁄ TMP Both mutations have the
potential to directly affect binding, and may do so
in a way that prohibits the other mutation
His45Arg
As expected from its location within the
NADPH-binding pocket, this mutation has a marked effect
on the binding of NADPH The cause of the
accom-panying increase in TMP resistance is unclear,
parti-cularly as DHF binding is relatively unaffected The
likely explanation is that the binding conformation
of NADPH is altered in such a way to favour DHF
binding over TMP
Ile943Leu
These residues form part of the DHF⁄ TMP-binding
pocket, and although they have no effect on the
polarity of the pocket, both cause steric changes
cap-able of favouring DHF over TMP due to the
observed difference in binding conformations of the
two ligands [7] (Fig 3) This change has only a
mediocre effect on TMP resistance, combined with
the loss of DHF and NADPH binding means that this mutation, like M20V, is only favoured at high concentrations of TMP
Lys109Arg Unfortunately, the insolubility of this point mutant prevents kinetic data from being collected, however, previous work [8] has shown that the two DHFR sub-units move relative to each other during the catalytic cycle Residue 109 is located on the ‘hinge’ between the two subunits and it is likely that the effect of any mutation in this region will be due to an effect on this movement The insolubility caused by this mutation is
a significant evolutionary cost, and may serve to explain why it is only observed in later rounds Other mutations acquired in the directed evolution process must be responsible for restoring the solubility of the enzyme
Concluding remarks The initial aim of this study was to identify residues that could be mutated to reduce the affinity of E coli DHFR for TMP We did not investigate the mecha-nisms by which mutations could arise in the clinical environment, our aim was to better understanding of how the active site could mutate in response to an anti-biotic while still maintaining activity Such information would aid in the design of new drugs Unfortunately, the picture that emerges in the case of DHFR is that, not only can mutations occur to overcome the effects
Fig 3 Binding of DHF and TMP by DHFR This illustrates the dif-ferences in crystallographically observed binding of the substrate and inhibitor Native residues are shown in green, mutants in red NADPH is shown in orange, DHF in gold and TMP in pink.
Trang 8of TMP with little loss of activity, but the ability to
increase that activity is also present Indeed, the
increase in DHFR activity is one mechanism used to
reduce the effect of TMP However, it may be possible
to screen mutants as well as native enzyme forms
when searching for new drugs, allowing for the
selec-tion of drugs effective against forms resistant to
cur-rent antibiotics
The similarities between some the mutations
pro-duced in this study and those found in clinical isolates
of TMP-resistant DHFR illustrate the power of
direc-ted evolution protocols to mirror the evolutionary
pro-cesses of nature, and add weight to its use as a tool
capable of predicting future developments in
antibacte-rial resistance
A trend apparent in our results is that the increase
in TMP resistance observed in the mutants occurs
without any accompanying loss in activity Although
the improvements in activity accompanying the
devel-opment of resistance run contrary to the normal
bene-fit⁄ cost trade-off expected of resistance development, it
can be understood in terms of evolutionary pressure
In the situation in which the reaction catalysed by an
enzyme is not a rate-limiting step in the growth and
reproduction of an organism, then there is no further
evolutionary pressure on that enzyme In such a case,
evolution halts even if the enzyme is still far from
opti-mized This results in an untapped pool of
evolution-ary potential, which remains available for future use
when the environment changes
Introduction of an antibiotic, in this case TMP,
results in a change in evolutionary pressure as the target
of the antibiotic (DHFR in this study) becomes the
cata-lyst for a reaction that is now limiting the growth of the
organism This prompts further evolution of the
previ-ously suboptimized enzyme, selecting not only for
resist-ance to the antibiotic, but also for improved substrate
binding and maximum activity Enzymes (such as
DHFR) possessing such a range of unused evolutionary
potential will make poor choices for antibiotic targets,
as it is relatively easy for the target enzyme to develop
not only resistance to the antibiotic, but increased
enzyme efficiency as well Although the mechanisms
involved in the spread of antibiotic resistance are not
part of this study, it is logical to conclude that mutations
that confer resistance without a drop in activity face
fewer barriers to their propagation and transfer than
genes that can only confer resistance with an associated
cost More highly evolved enzymes that are at or near
their evolutionary limit would be better targets for
anti-biotics Resistant mutants of such enzymes should have
lower activities and should disappear from the
popula-tion once the selecpopula-tion pressure is removed
An interesting point to note with respect to DHFR is that bacteria are capable of synthesizing folate and have
a great range of variation in DHFR genes Mammals are incapable of synthesizing folate and rely more hea-vily on cycling of dietary folate The increased import-ance of DHFR and other folate cycling enzymes to mammals may explain the fact that mammalian DHFRs are far more tightly conserved than bacterial ones [19] Compounds such as the chemotherapy drug methot-rexate, possessing higher affinity for E coli DHFR may allow for the collection of more information regarding active site mutations through further direc-ted evolution experiments Such work would require changes to the expression system, as E coli already possesses an innate resistance to methotrexate by means of an efflux system [20]
This study has shown the usefulness of directed evo-lution in drug design, particularly in the selection of drug targets Along with a clear indication of the ease with which antibiotic resistance can develop and the means by which it can do so, directed evolution can also provide a pool of resistant mutant proteins that may be useful in the screening of new drugs
Experimental procedures
Materials Enzymes used this research were obtained from New Eng-land Biolabs (Ipswich, MA), Roche (Basel, SwitzerEng-land) and Stratagene (La Jolla, CA) All other compounds were obtained from Sigma (St Louis, MO)
Isolation of E coli folA The E coli DHFR gene (folA) was PCR amplified from the chromosomal DNA of E coli strain DH5a The primers used for this were 5¢-CGCGCATGCCATATGATCAGTCTG ATTGCG, 3¢-CCAGGCCTGCATGCTTACCGCCGCTC CAGAATCTC The gene was cloned into pCL476 using the restriction enzymes NdeI and SphI pCL476 [21] is a heat-inducible vector containing an ampicillin (Amp) resistance marker and a six-histidine tag at the N-terminal end of the expressed gene This vector was used for all rounds of direc-ted evolution
When protein was purified from the pCL476 vector, although this protein was active, it failed to crystallize under previously published conditions The expression vector was then changed to one without the six-histidine tag, pJWL1030 [22] This protein was eventually crystallized under condi-tions similar, though not identical to published condicondi-tions Genes were amplified using the primers 5¢-CGCGCATGC CATATGATCAGTCTGATTGCG, 3¢-CCCAAGCTTCTG
Trang 9CAGTTACCGCCGCTCCAG, then cloned into pJWL1030
using the restriction enzymes NdeI and PstI pJWL1030 is
constructed from pJJkm and pCY476, and contains a
kana-mycin (Kan) resistance marker The inserted gene is
expressed using a constitutive LacZ operon
Assays of enzyme expression levels and MIC tests were
performed using pCL476 as the vector, all other assays
used enzyme expressed using pJWL1030
Expression and purification
All experiments were undertaken at 4C unless otherwise
stated The pJWL1030:folA construct was transformed in
to competent E coli DH5a cells, and grown at 37C to an
optical density of 0.6–0.7 Purification is based on that used
by Shaw et al [23], and only a brief description will be
given here Cells were harvested and lysed on a French
press in 0.1 mm Tris buffer (pH 7.5) containing 10 lm
2-mercaptoethanol (buffer A) The soluble faction was loaded
onto a Q Sepharose HP Affinity Column (GE Healthcare,
Chalfont St Giles, UK), and eluted with a similar buffer
containing 0.5 m KCl (buffer B) Elution occurred at
0.25 m KCl, confirmed by SDS ⁄ PAGE Factions
contain-ing DHFR were loaded onto a Sephandex G75
size-exclu-sion column (GE Healthcare) and eluted with buffer A
The resulting protein was concentrated to between 20 and
50 mgÆmL)1 and stored at 4C Yields were typically 30–
40 mg per 1 L of culture Yields of mutant enzymes were
lower than that of the native form, ranging between 80 and
90% of the amount of DHFR obtained per 1 L of culture
Shuffling
The DNA shuffling method described by Stemmer [24] was
used to introduce random mutations in to the folA gene
The folA gene was first amplified by PCR using the same
primers as above Amplification was conducted using Taq
DNA polymerase to allow for the introduction of
transcrip-tion errors The PCR product was purified then digested
using DNase I to produce fragments with an average length
of 100 bp Fragments between 50 and 150 bp in length were
purified using gel electrophoresis These fragments were
then reassembled by primerless PCR, then amplified using
the above primers for pCL476 and inserted into the
expres-sion vector using NdeI and SphI The mutagenesis rate of
this protocol has been previously established at 0.7% [24]
Selection
The mutant library generated was transformed into
compet-ent DH5a cells and plated onto Mueller Hinton agar
(MHA) plates containing Amp (50 mgÆL)1) and TMP
Inhi-bition of thymidine production is a major aspect of the
activity of TMP, and inclusion of thymidine in the media
allows bacterial growth regardless of the amount of TMP present This necessitated the use of the specialist media MHA, as Luria–Bertani medium contains thymidine Plates were incubated at 37C for 24–48 h and 200 colonies were selected for secondary screening, based on colony size, with the largest colonies being selected These colonies were grown in minimal A medium (MMA) [25] containing Amp and TMP (concentrations as below) overnight The D595of each was measured as an indicator of growth rate and the
20 colonies with the highest D595selected for DNA sequen-cing Following sequencing, the best (as determined by the measured growth rate) 10–15 unique mutants were used to generate the next round of mutants The first round of evo-lution was selected using media containing 2 mgÆL)1TMP, the second with 10 mgÆL)1 TMP, the third with 50 mgÆL)1 TMP and the fourth with media containing 100 mgÆL)1 TMP The TMP concentrations used in the third and fourth rounds are comparable with MIC values of clinically isolated TMP-resistant strains of E coli [26]
A number of amino acids were selected for site-specific mutagenesis based on trends seen in the characterization of round three and four directed evolution mutants, and in mutation frequency as seen in Table 1
Point mutant generation Single-site mutants were generated using point mutagenesis Primers 33 bp in length were designed to be homologous to the section of the folA gene containing the base to be mutated, excepting the centre three amino acids that code for the new base The folA gene inserted in the pJWL1030 vector was subjected to PCR using these primers and then digested with DpnI, removing the original template DpnI is used for this purpose as it will only digest DNA that has been methylated The template DNA, having been isolated
in plasmid form from E coli, is methylated, but the newly synthesized (and shuffled) DNA is not
Kinetic assays Due to the low concentrations of DHF required to avoid substrate inhibition (first noted by Stone et al [16]) in these assays, NADPH fluorescence (ex 340, em 465) was used to increase sensitivity This substrate inhibition is not expected
to be an issue under normal physiological conditions, as it
is only observed for concentrations of DHF in excess of
1 lm and normal intracellular concentrations of DHF are
< 28 nm [27] All fluorescence measurements were made on
a Varian Cary Eclipse Fluorescence Spectrometer The reac-tion was monitored by measuring the drop in NADPH fluorescence as the reaction progressed DHFR (3.7 nm) was preincubated with DHF (0.2–0.8 lm) for 30 s in reac-tion buffer (0.1 m KHPO4pH 7.0, 100 mm 2-mercaptoetha-nol) based on that originally described by Baccanari et al
Trang 10[14] The preincubation was required to avoid the hysteretic
behaviour of E coli DHFR [28] The reaction was initiated
by the addition of NADPH (0.2–1.0 lm) and monitored to
completion Initial rates were recorded and used to
calcu-late the Michaelis–Menton constants
Binding assays
Binding affinity of TMP for the enzymes was measured by
monitoring the quenching of tryptophan absorbance of the
enzyme (ex 280 nm, em 345 nm) as increasing amounts of
TMP were titrated in Measurements were made in reaction
buffer (0.1 m KHPO4 pH 7.0, 100 mm 2-mercaptoethanol)
with DHFR (50–200 nm) TMP was titrated at
concentra-tions to ensure that no more than 5 lL was added at a time
The reaction was allowed 2 min to equilibrate after each
addition Fluorescence of TMP was measured by use of a
parallel reaction in which the DHFR was replaced by enough
Trp to give an equivalent fluorescence and used to correct the
final readings Binding constants were calculated by fitting
data to equations 1 and 2 as according to Stone et al [16]
F¼ ½F0Et F1ðELÞ=Et ð1Þ
Kd¼ ðEt ELÞðLt ELÞ=ðELÞ ð2Þ
where F, F0, and F¥are the observed florescence, the
flores-cence of free enzyme and the floresflores-cence of the enzyme
ligand complex, respectively; Lt, EL, and Etare the
concen-trations of total ligand, the enzyme–ligand complex, and
the total enzyme, respectively kaleidagraph was used to
fit the data using nonlinear regression, in all cases giving a
good fit with R-values in excess of 95%
Stability assays
Heat stability of the enzyme was determined by incubating
aliquots of the enzyme at (45–65C) for 30 min, then
assay-ing for activity as described above at concentrations of
0.8 nm DHF and 0.8 nm NADPH Residual activity was
measured by calculating the proportion of initial velocity
remaining after heating as a proportion of the initial
velo-city of the nonincubated enzyme
MIC assays
To determine the MIC for TMP for each mutant evolved,
transformants were grown overnight on MHA plates
con-taining amounts of TMP varying from 0 to 200 mgÆmL)1in
steps of 50 mgÆmL)1
Acknowledgements
The authors thank Cameron McRae of the
Bimolecu-lar Resource Facility for DNA sequencing
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