Characterization of substrate binding and enzymatic removal of a 3-methyladenine lesion from genomic DNA with TAG of MDR A. baumannii

Article abstract of DOI:10.1039/c6mb00517a

The rise of multiple-drug resistance in bacterial pathogens imposes a serious public health concern and has led to increased interest in studying various pathways as well as enzymes. Different DNA glycosylases collaborate during bacterial infection and disease by overcoming the effects of ROS- and RNS-mediated host innate immunity response. 3-Methyladenine DNA glycosylase I, an essential DNA repair enzyme, was chosen for the present study from the MDR species of A. baumannii. The enzyme was especially chosen because of its functional significance in A. baumannii and due to its structural variation from its human homologue. MDR strains such as A. baumannii are interesting targets owing to their evolved mechanisms of evading a host defence. In the absence of any structural information, the enzyme was characterized biophysically and biochemically. Binding studies with 3mA and Zn²⁺ indicated that the activity of TAG-Ab is an enthalpy-driven process. Fluorescence thermal denaturation studies described that the denaturation of TAG-Ab is a two-step process. Modified RP-HPLC-based glycosylase assay attested that the heterologously expressed and purified TAG-Ab enzyme is active and catalyses the removal of 3mA. Other binding parameters and the effect of adenine on substrate binding are also discussed in detail.

Full text of DOI:10.1039/c6mb00517a

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Characterization of substrate binding and
enzymatic removal of a 3-methyladenine
lesion from genomic DNA with TAG of
MDR A. baumannii†
Cite this:DOI: 10.1039/c6mb00517a
Received 8th July 2016,
Accepted 7th September 2016
Jyoti Singh Tomar,a Manju Narwal,b Pravindra Kumarb and Rama Krishna Peddinti*a
DOI: 10.1039/c6mb00517a
www.rsc.org/molecularbiosystems
The rise of multiple-drug resistance in bacterial pathogens imposes and it is known to mainly target the nucleobases guanine and
a serious public health concern and has led to increased interest adenine.2–4 SAM generates 3-methyladenine (3mA) and 7-methyl-
in studying various pathways as well as enzymes. Different DNA guanine (7mG) lesions by an SN2 mechanism. Here, 7mG
glycosylases collaborate during bacterial infection and disease by is mildly deleterious, while 3mA is strongly detrimental and
overcoming the effects of ROS- and RNS-mediated host innate effectively blocks the action of polymerase enzymes, resulting in
immunity response. 3-Methyladenine DNA glycosylase I, an essen- a strong cytotoxic effect.5 Every cell has a range of repair enzymes
tial DNA repair enzyme, was chosen for the present study from the that are expressed to remove such modifications from the DNA.
MDR species of A. baumannii. The enzyme was especially chosen In E. coli, two different DNA glycosylases, namely 3-methyladenine
because of its functional significance in A. baumannii and due to its DNA glycosylase I (TAG) and 3-methyladenine DNA glycosylase II
structural variation from its human homologue. MDR strains such (3MAG), have been found to excise alkylated lesions present in the
as A. baumannii are interesting targets owing to their evolved DNA. However, in humans, the 3-alkyladenine DNA glycosylase
mechanisms of evading a host defence. In the absence of any (AAG) enzyme is responsible for the base excision repair of 3mA
structural information, the enzyme was characterized biophysically and many other alkyl lesions.6,7 As the structure and function of
and biochemically. Binding studies with 3mA and Zn2+ indicated that human DNA glycosylase AAG is entirely different from prokaryotic
the activity of TAG-Ab is an enthalpy-driven process. Fluorescence and lower eukaryotic DNA glycosylase enzymes, it makes TAG and
thermal denaturation studies described that the denaturation of 3MAG selective targets for researchers. Recently, Tang and
TAG-Ab is a two-step process. Modified RP-HPLC-based glycosylase co-workers stated that both intracellular and extracellular patho-
assay attested that the heterologously expressed and purified gens must survive the onslaught of the host innate immune system
TAG-Ab enzyme is active and catalyses the removal of 3mA. Other during colonization and disease spreading.8 A key component of
binding parameters and the effect of adenine on substrate binding the innate immune response is the generation of reactive oxygen
are also discussed in detail.
species (ROS) and nitrogen species (RNS) by phagocytic cells, which
target and disrupt pathogen DNA. The overexpression of DNA
glycosylases is the most important mechanism for the repair of
such ROS/RNS-mediated DNA damage. DNA damage in pathogenic
bacteria occurs when they are exposed to these ROS and RNS via
oxidation, deamination and alkylation. To overcome this damaging
effect of host immunity, these pathogens express a large number of
DNA glycosylases that recognize and excise away the damaged
nucleobases. This way, DNA glycosylases find significance in the
survival of these pathogens against innate immunity.
Recently, studies performed on MutY by David and co-workers
inferred the importance of the zinc motif in the damage recogni-
tion and base excision mechanism.9 However, the zinc motif in
TAG is known to stabilize helix-hairpin-helix (HhH) fold.10 Various
studies on the enzyme TAG show that the presence of a zinc motif
is crucial for its structural stability as well as for the proper
functioning of TAG. To the best of our knowledge, no thermo-
Introduction
Genomic DNA acts as a blueprint for life allowing offspring to
inherit the characteristics of their parents. Initially, DNA was
imagined to be highly stable up until 1993, when Lindahl
refuted this fact by outlining the instability of DNA.1 Further
studies by different researchers established that cellular DNA is
constantly damaged by various processes, such as hydrolysis,
oxidation and non-enzymatic methylation. The most common
endogenous alkylating agent is (S)-adenosylmethionine (SAM)
a Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee-247667,
India. E-mail: rkpedfcy@iitr.ac.in, ramakpeddinti@gmail.com
b Department of Biotechnology, Indian Institute of Technology Roorkee,
Roorkee-247667, India
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6mb00517a dynamic data and binding parameters of Zn2+ with the zinc motif
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are available in the case of TAG from any bacterial sps. Thus, isothermal titration calorimetry (ITC) assay was performed
the TAG-Ab enzyme from A. baumannii was cloned, hetero- using an ITC-200 Microcal calorimeter (GE Healthcare). The
logously expressed and purified in 25 mM Tris buffer using a reaction cell was filled with 200 mL protein (500 mM) in 25 mM
two-step purification process. The recombinant TAG-Ab was Tris buffer and the syringe was loaded with 40 mL of each
found to maintain its 3D conformation and structural integrity substrate (3mA: 0.5 mM, ZnCl2: 0.3 mM) as the binding partner.
during the purification procedure.11 TAG exhibits little sequence About 20 injections of 2 mL of each substrate were performed
similarity with members (AlkA, EndoIII, hOGG1 and MutY) of the with a stirring speed of 2000 rpm. The time interval between
HhH superfamily; however, structurally it consists of a similar two injections was set to 120 s to ensure optimum mixing and
HhH domain. The present study was performed to elucidate the accurate measurement of the change in enthalpy. The titration
effect of thermal denaturation on the structure of TAG-Ab. The data of the ligand with protein-free buffer was used as a reference.
thermodynamic and binding parameters of Zn2+ with the zinc The data analysis was done using one binding site model in the
motif have not yet been studied. Therefore, to shed some light on Origin software version 7.0.
the strength and nature of binding with Zn2+, 3mA and adenine,
RP-HPLC-based glycosylase assay
ITC and intrinsic fluorescence experiments were performed here.
Also, a modified glycosylase assay was developed to confirm the
activity of the TAG-Ab enzyme. The significance of the TAG-Ab
enzyme lies in the fact that it is crucial for the survival of
A. baumannii. It shares sequence as well as structural homology
with the TAG of S. typhi and of many other pathogenic bacteria
but it is considerably different from the human alkyl glycosylase
enzyme (ESI,† Fig. S1).
A Shimadzu Inc. (Japan) HPLC system, attached with an ultra
violet detector, was used for the experiments.14 The chromato-
graphic separations were carried out on a C18 column under
isocratic conditions with a flow-rate of 0.5 mL minꢁ1. The mobile
phase was composed of an acetonitrile and water (85 : 15) mixture.
Each component of the mobile phase was degassed before
use. The wavelength range of the diode array detector was set
to 220–360 nm and the injection volume was set to 20 mL.
Then, 50 mM of methylated genomic DNA (cellular methylated
DNA) was incubated with TAG-Ab enzyme (3 mg mLꢁ1) and the
amount of released free 3mA as a product was quantified from
Materials and methods
Spectrofluorimetric titration studies
Fluorescence spectra were recorded on a Fluorolog-3 Spectro- the area under the peak. The above mixture was incubated at
fluorimeter LS55 (make HORIBA Jobin Yvon Spexs). The 4 1C and 37 1C for varying amounts of time. To bring the reaction
intrinsic fluorescence emission spectrum in the range of to an end, the reaction mixture was subjected to neutral thermal
290–550 nm was recorded upon excitation at 280 nm. Slit widths hydrolysis at 50 1C. Then, cold ethanol was used to precipitate
of 5 nm and a sample volume of 1 mL were used and the depurinated DNA and the supernatant was subjected to HPLC
concentration of protein was adjusted to obtain a satisfactory analysis. Calibration of the HPLC curve was performed with a
signal to noise ratio. Thermal denaturation was performed to standard solution of 3mA at a concentration of 0.5 mg mLꢁ1. The
observe the effect of temperature (20–75 1C) on the TAG-Ab control DNA and TAG-Ab enzyme were also analyzed under the
enzyme in 25 mM Tris buffer. For the denaturation measure- same experimental conditions to calculate their retention times.
ments, sample cells were equilibrated for 90 s at each tempera-
ture. The binding affinity of 3mA with TAG-Ab was determined
by following the decrease in tryptophan fluorescence. TAG-Ab with
a fixed concentration was titrated against increasing amounts of
3mA (1–500 mM) at 20 1C. In order to determine the dissociation
Results and discussion
Denaturation and substrate binding studies by fluorescence
Intrinsic tryptophan fluorescence is very sensitive to the local
electrostatic environment and hence it is considered as a key
probe for exploring the conformational changes of proteins
in solution. Many studies conclude that the fluorescence wave-
constant Kd, the binding data was fitted with the following
equation using the program Sigma Plot.12
1
F ¼ f1 ꢀ Po þ ð f2 ꢁ f1Þ½ðPo þ Lo þ KDiss
Þ
2
length maximum of a tryptophan is determined by the local
electric field projection along the long axis of the indole ring.15
The interactions, such as conformational transitions, subunit
association, substrate binding or the denaturation affect the
local environment and can be monitored by using the trypto-
phan emission spectrum. The structural integrity of TAG-Ab has
been previously verified by far-UV CD experiments;11 therefore in
this study, thermal denaturation experiments were performed
and the denaturation curve plotted as the intrinsic fluorescence
intensity ratio F350/F330 vs. temperature (Fig. 1A). The emission
spectrum of tryptophan depends on the solvent polarity as well
(1)
ꢀ
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
ꢁ
ðPo þ Lo þ KDissÞ ꢁ4 ꢀ Po ꢀ Lo
where f1 denotes the fluorescence signal of the protein with
zero concentration of substrate and f2 is the fluorescence signal
of the protein when it is fully saturated. P0 is the concentration
of protein, L0 is the concentration of the substrate and KDiss is
the binding constant.13
Calorimetric characterization of TAG-Ab substrate binding
To determine the binding parameters of TAG-Ab with the as on the interaction of the solvent with its imino group.
substrates 3-methyladenine, adenine and zinc chloride, an Tryptophan forms a hydrogen bond with water, which results
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was used to estimate the Kd. The calculated Kd value of 3mA
binding was 246 mM, which is comparable with the TAG of
S. aureus [220 mM],17 while it is higher than that from the TAG
of E. coli [68 mM].18 The titration experiments of TAG-Ab with
3mA, performed in the presence of adenine, did not exhibit any
effect on the substrate binding (ESI,† Fig. S2C). The titration of
the TAG-Ab enzyme with adenine alone displayed no change
in the emission spectra, thus indicating the binding selectivity
of the TAG-Ab enzyme.
Isothermal titration calorimetry binding assay of TAG-Ab with
3mA and Zn2+
ITC is a reliable and widely used method to evaluate the energetics
of macromolecular interactions by recording a thermogram
of differential heating powers during a series of injections.
The changes in molar enthalpy DH1 and in the molar Gibbs
free energy DG1 (or, equivalently, the equilibrium dissociation
constant Kd or the association constant Ka = 1/Kd), as well as the
stoichiometry (n) of the reaction can be deduced from the
shape of the saturation curve, which is obtained in the form
of a titration isotherm of the successive reaction heating. From
a single ITC experiment, a detailed thermodynamic decomposi-
tion of driving forces in terms of the thermodynamic para-
meters DH1, DH1–TDS1 = DG1 and DG1 = RT ln(Kd/M) can be
obtained.
Fig. 1 (A) Thermal denaturation of TAG monitored at
a wavelength
Here, ITC was employed to investigate the thermodynamic
nature of the TAG-Ab DNA repair enzyme association with its
substrate 3mA, and with adenine plus the metal Zn2+. In 2012,
Naismith and co-workers performed ITC experiments to
monitor the effect of pH on the binding strength of the 3mA
with the enzyme TAG-S. aureus.17 However, the thermodynamic
parameters as well as the stoichiometry of the binding were not
determined. TAG is known to have specificity towards 3mA
of 350 nM and the change in the intrinsic fluorescence intensity ratio
350/F330 plotted at different temperatures; the curve was fitted to the data
F
assuming a two-state unfolding. (B) Quenching of the intrinsic fluorescence
of the TAG enzyme on binding with 3mA at a low protein concentration.
TAG shows B65% decrease in fluorescence intensity; the square box
represent the average fluorescence; Kd for 3mA determined from these
data is 246 mM.
in a shift of the emission spectrum to a longer wavelength (lmax binding unlike AAG or other AlkA enzymes, which have a broad
from B330 to 350 nm). Due to this reason, the fluorescence substrate range. To elucidate the parameters governing the
intensity ratio F350/F330 is widely used as a probe for monitoring binding of TAG-Ab with its substrate 3mA, titration studies were
the denaturation of proteins. The TAG-Ab fluorescence emis- done. The upper panel of Fig. 2 depicts the heat exchanged per
sion spectrum, recorded as a function of temperature, demon- injection of 3mA into the TAG-Ab solution, while the lower panel
strated that the protein was stable up to 35 1C. However, represents the integrated data and the non-linear least-squares
on further heating, protein denaturation started (Fig. 1A, ESI,† fit to a single site-binding model. The obtained ITC profile for
Fig. S2A) and 50% denaturation of the TAG-Ab was completed at 3mA is characterized by a stoichiometric ratio of one protein
about 55 1C. Heating the protein sample above 65 1C did not molecule per 3mA. The binding of 3mA to TAG-Ab is an enthalpy-
show any measurable difference in the fluorescence intensity, driven process (DH = ꢁ12.6 ꢂ 0.4 kcal molꢁ1). The dissociation
which may be attributed to its complete denaturation. The constant Kd = 266 ꢂ 0.2 mM (Fig. 2), is also comparable with the
TAG-Ab denaturation curve exhibited an apparently cooperative binding constant obtained from fluorescence experiments.17
single transition between its folded and an unfolded state,
The binding of 3mA substrate with TAG-Ab is not favoured
thus confirming the denaturation of the TAG-Ab as a two-step by the entropy term (ꢁTDS = 9.1 kcal molꢁ1). However, it might
process.16
be explained as: the binding site of TAG-Ab is filled with water
Titration experiments were performed to calculate the sub- molecules, which might get relocated upon binding of the
strate 3mA binding parameters at pH 7.0 using eqn (1), as ligand, concurrently producing a positive entropy contribution
described in the methodology section. Titration of the TAG-Ab to DG.11,19 The DG value, calculated using the above entropy and
enzyme with the 3mA substrate resulted in a B77% decrease of enthalpy values, was found to be ꢁ3.5 kcal molꢁ1, indicating
intrinsic fluorescence intensity (Fig. 1B, ESI,† Fig. S2B), unlike that the binding of 3mA is an exothermic process. The binding
the titration of the TAG of E. coli, in which titration with the stoichiometry (n) is 0.934, meaning one monomer of TAG-Ab
substrate resulted in a 45% decrease in the fluorescence intensity. binds one molecule of 3mA. The binding affinity of alkA enzyme
The binding of 3mA quenches tryptophan fluorescence, which is nM range, unlike TAG, which has a binding in the mM range,
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the alkylpurine DNA glycosylases can be characterized by the
radioactive detection of alkyl bases liberated from genomic DNA,
but this is costly and poses an environmental hazard. Indeed,
the involvement of radio-labelled nucleobases, especially in the
case of alkylation adducts, is a laborious, costly and toxic
task.22–25 There are two widely used methods for performing
the glycosylase assays for monitoring the TAG base excision
activity; the first method involves the liberation of a single,
defined lesion that has been chemically or enzymatically incor-
porated into an oligonucleotide.26–28 This method is good for
the characterization of stable lesions; however, it cannot be
performed for the modifications (N3- and N7-substituted alkyl-
purines), which are prone to spontaneous depurination or ring
opening under the conditions required for oligonucleotide
synthesis and purification.12,29 The second method involves
the excision of nucleobases from genomic DNA that has been
pre-treated with an alkylating agent, followed by ethanol pre-
cipitation of the DNA and HPLC separation of the soluble
fraction. This method has an advantage of allowing multiple
types of alkylated bases to be evaluated and has also been found
Fig. 2 ITC binding isotherm for the association of TAG with substrate
3mA at 20 1C. The upper panels correspond to the total heat exchanged to be useful for characterizing labile lesions (3mA). However,
upon injecting the aliquots of substrate. The lower panels show the
in this method, glycosylase activity of the alkylpurine DNA
glycosylases requires radioactive labeling of alkyl bases which
after enzymatic cleavage generates non eco-friendly by-products.
resultant binding isotherms acquired by integrating the peak areas of each
injection. The continuous line represents the non-linear least-squares
fitting of the affinity (Ka) and the change in enthalpy DH and entropy DS
In the present work, a second methodology with minor
modifications was applied to detect the methylated nucleo-
bases liberated from the control genomic DNA as a result of
the DNA glycosylase activity of the TAG-Ab enzyme. Time-
dependent HPLC analysis of the reaction mixture was done to
monitor the liberation of free 3mA qualitatively. First, the
control DNA was subjected to analysis and a peak at a retention
time of 2.93 min for the control DNA was obtained (Fig. 3A).
HPLC analysis of TAG-Ab (as positive control) produced three
elution peaks, at 4.13 min for TAG-Ab (major), 3.2 min (minor)
and 8.18 (minor) min (Fig. 3B). The minor peaks might be due
to some impurity present in the protein sample.
to a single-site-binding model.
showing that AlkA has 1000 times higher affinity than TAG.
However, similar to TAG, the binding process in the case of
AlkA is largely enthalpy-driven and it counteracts the entropic
contributions, resulting from a high degree of ordering of the
enzyme in the DNA-bound complex. We also performed the
titration of TAG-Ab with adenine, but it did not exhibit any
measurable exchange of heat, designating that adenine does
not bind with the enzyme (ESI,† Fig. S3A).
Analysis of the ITC profile of TAG-Ab with zinc indicated a
DG value of ꢁ4.7 kcal molꢁ1 and an n value of 3.8 (ESI,†
Fig. S3B). It is known from the literature20,21 that Zn2+ pos-
sesses one binding site on TAG; however, an n value of 3.8 in
the case of TAG-Ab indicates a higher number of binding sites,
which could be because of the nonspecific binding. Due to this,
it is difficult to infer anything from the ITC data. Stivers and
co-workers20 found that the high binding affinity of Zn2+ might
be due to its indirect role in organizing the structure of the active
site and the tethering of the unstructured N- and C-terminal
regions of the enzyme, which mimics the functional role of the
protein–protein interactions present in the larger HhH super-
family members. The binding of zinc with TAG-S. typhi includes
4 H-bonded and 12 non-bonded interactions with the Cys4,
His17, His175 and Cys179 residues. The distance between the
active site and zinc motif is B10 Å (ESI,† Fig. S3C).
In the study done by Eichman and co-workers,30 it was
found that at 37 1C, the process of depurination of 3mA from
the DNA is completed after 30 min. Similarly, we found that the
new peak emerging at 12.5 min became constant after 30 min
(ESI,† Fig. S4). Since it was observed in the fluorescence studies
that the denaturation of TAG-Ab starts at 35 1C, we thus
performed the enzyme assay at a lower temperature of 4 1C.
The TAG-Ab was incubated for 15 min with the substrate
molecules as described in the methodology section, whereby
20 mL of reaction mixture was loaded on a C-18 column. TAG-Ab
was incubated with substrate molecules for different time
intervals. Initially, the runtime for the control sample was set
to 45 min, but since no peak was observed after 15 min of run,
we thus set the runtime for 15 min for the subsequent runs. After
injecting, the reaction sample peak was observed emerging at
12.5 min after injecting on the column. In the subsequent
injections at different time intervals for the incubated samples
DNA glycosylase assay using HPLC
The detection of free nucleobases released from genomic of TAG-Ab with the substrate, the area and the intensity of this
DNA traditionally involves HPLC separation and scintillation peak at 12.5 min was observed to increase (Fig. 3C and D).
detection. The glycosylase activity and substrate preferences of The area and intensity of the peak was observed to increase
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Fig. 3 Excision of 3mA from the control genomic DNA by TAG of A. baumannii using RP-HPLC. (A) Chromatogram of the control genomic DNA.
(B) Chromatogram of the TAG protein. (C) TAG excised 3mA peak emerged after 20 min and (D) after 100 min incubation time. (E) Chromatogram of 3mA
for the reference. Methylated genomic DNA was incubated with TAG for 120 min at 4 1C; 20 mL supernatant of reaction mixture was injected into the
HPLC column. Time dependence of 3mA excision by TAG appeared at B12.5 min.
up to 100 min incubation time of the TAG-Ab/substrate reaction complementarity and hydrogen bonds. Tryptophan side chain
mixture. The peak intensity and area did not change on further participates in the p–p interaction with the aromatic ring of
incubation, which could be either because the substrate was 3mA, correspondingly we have found large decrease in the
consumed completely or the enzyme was saturated with the intrinsic fluorescence spectrum on titrating with the 3mA
substrate (ESI,† Fig. S5, Table S1). To reconfirm that the new substrate. The obtained biophysical results are in line with
peak corresponded to the expected reaction product, the the literature data18 and our theoretical results.11
standard solution of 3mA was analyzed and found to be the same
The main scientific challenge in developing inhibitors of
(12.64 min) (Fig. 3E). This showed that the enzyme was active at DNA repair is target specificity.31 As we already discussed, TAG
low temperature (4 1C); however, the activity was considerably low. possesses a significant difference in sequence as well as in
This is the first report on functional studies of the TAG-Ab enzyme structure from the human alkyl DNA repair enzyme (AAG),
from A. baumannii action at low temperature, and the glycosylase which makes it a useful selective target. Its thermodynamic
assay performed here clearly indicated that the recombinant parameters and thermal stability alongside a simple method to
TAG-Ab enzyme was active.
examine enzyme activity can be utilized in drug discovery to
Binding of substrate 3mA imposes some conformational gain an insight into the binding modality and identification of
changes in enzyme as we have found from the CD spectroscopy thermodynamically privileged chemical scaffolds. Described
result,11 also binding of 3mA involves hydrogen bonding, p–p herein are some biophysical approaches that can complement
interaction and van der Waals interactions with the active site the measures of various aspects of the substrate binding and
residues. 3mA builds two hydrogen bonds with residues Tyr15 can facilitate the discovery of competitive TAG inhibitors. For
(2.6 Å) and Tyr18 (2.9 Å) of the TAG, respectively. It is known example, ITC covers the changes in both enthalpy and entropy,
fact that generally favourable change in the enthalpy value is where a favourable change in enthalpy value is basically driven
achieved by formation of bonding networks mediated by shape by specific bonding networks mediated by hydrogen bonds,
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shape complementarity and salt bridges; however, positive entro- Acknowledgements
pic contributions are derived from hydrophobic interactions or
JST thanks MHRD and MN thanks UGC, New Delhi for research
fellowships. We thank Vijay Sharma for his assistance in
performing ITC experiments.
an increase in the conformational flexibility on complexation.
A growing body of evidence indicates that thermodynamic studies
should be harnessed early in drug discovery to differentiate
chemical scaffolds based on their enthalpic efficiency.19 Rojas
and co-workers inferred that thermal stability provides more
valuable information than EC50 values while differentiating and
analyzing inhibitory compounds. Finally, an easy enzymatic assay
could be utilized to see the effects of substrate-based competitive
inhibitors11 on enzyme activity. Collectively, the experimental
approaches described herein and complementary computational
studies32 may facilitate the discovery of competitive inhibitors
that could end in anti A. baumannii molecules.
Notes and references
1 T. Lindahl, Instability and decay of the primary structure of
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2 R. de Bont and N. van Larebeke, Endogenous DNA damage
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3 B. Sedgwick, Nitrosated peptides and polyamines as endo-
genous mutagens in O6-methylguanine-DNA alkyltransferase
deficient cells, Carcinogenesis, 1997, 18, 1561–1567.
4 B. Rydberg and T. Lindahl, Nonenzymatic methylation of
DNA by the intracellular methyl group donor S-adenosyl-l-
methionine is a potentially mutagenic reaction, EMBO J.,
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5 T. O. Eloranta, Tissue distribution of S-adenosylmethionine
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6 T. Hollis, A. Lau and T. Ellenberger, Structural studies of
human alkyladenine glycosylase and E. coli 3-methyladenine
glycosylase, Mutat. Res., DNA Repair, 2000, 460, 201–210.
7 M. Hedglin and P. J. O’Brien, Human Alkyladenine DNA
Glycosylase Employs a Processive Search for DNA Damage,
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8 S. van der Veen and C. M. Tang, The BER necessities: the
repair of DNA damage in human-adapted bacterial patho-
gens, Nat. Rev. Microbiol., 2015, 13, 83–94.
9 L. M. Engstrom, M. K. Brinkmeyer, Y. Ha, A. G. Raetz,
B. Hedman, K. O. Hodgson, E. I. Solomon and S. S. David,
A zinc linchpin motif in the MUTYH glycosylase inter-
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Conclusions
TAG hydrolyzes the glycosidic bond of methylated adenine in DNA
in various bacteria and some eukaryotes. It exhibits less primary
sequence similarity with members of the HhH superfamily of DNA
glycosylases. Unlike other glycosylases, it has specificity for the
3mA base. Here, affinity purified TAG-Ab protein was used for the
stability and substrates binding analysis. The enzyme activity was
confirmed by the RP-HPLC-based DNA glycosylase assay. A protein
folding/unfolding study showed that the thermal denaturation of
TAG-Ab is a two-step process. The binding parameters of substrates
with the TAG-Ab enzyme revealed that the enzyme exhibits selec-
tivity for 3mA over the normal adenine base and that the enzyme
has high binding affinity for Zn2+ over 3mA. Analysis of the enzyme
activity assay indicated that a new peak, emerging at 12.5 min,
corresponded to the retention time of 3mA, attesting that the
enzyme is active. This study furnishes the thermodynamic para-
meters and binding characterization, thereby providing the basis
for an initial map of the energetic landscape of the damaged base
binding during damage repair in TAG-Ab. TAG exhibits binding
selectivity for 3mA over adenine, which is in line with the reported
results. Structural information is needed to establish the struc-
ture–function–energy correlations to understand the repair mecha-
nism completely. The information obtained from biophysical and 10 A. C. Drohat, K. K. Daniel, J. Krosky and J. T. Stivers,
biochemical experiments performed here is of importance in
designing competitive inhibitors.
3-methyladenine DNA glycosylase I is an unexpected helix-
hairpin-helix superfamily member, Nat. Struct. Biol., 2002,
9, 659–664.
11 J. S. Tomar and R. K. Peddinti, Optimized method for TAG
protein homology modeling: In silico and experimental
structural characterization, Int. J. Biol. Macromol., 2016,
88, 102–112.
12 The data was fit with a modified three parameter exponential
decay using SigmaPlot version 12.5, from Systat Software,
Inc., San Jose California USA, www.systatsoftware.com.
13 P. J. O’Brien and T. Ellenberger., The Escherichia coli
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Abbreviations
AAG
3mA
3-Alkyladenine DNA glycosylase
3-Methyladenine
3MAG
7mG
3-Methyladenine DNA glycosylase II
7-Methylguanine
RNS
ROS
Reactive nitrogen species
Reactive oxygen species
RP-HPLC
Reverse phase high performance liquid
chromatography
SAM
TAG
(S)-Adenosylmethionine
3-Methyladenine DNA glycosylase I
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