Enzymological and structural studies of the
mechanism of promiscuous substrate recognition by
the oxidative DNA repair enzyme AlkB
Bomina Yuaand John F. Hunta,b,1
aDepartment of Biological Sciences, 702A Fairchild Center, MC2434, andbNortheast Structural Genomics Consortium, Columbia University,
New York, NY 10027
Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved June 22, 2009 (received for review December 18, 2008)
Promiscuous substrate recognition, the ability to catalyze trans-
formations of chemically diverse compounds, is an evolutionarily
advantageous, but poorly understood phenomenon. The promis-
cuity of DNA repair enzymes is particularly important, because it
enables diverse kinds of damage to different nucleotide bases to
be repaired in a metabolically parsimonious manner. We present
enzymological and crystallographic studies of the mechanisms
underlying promiscuous substrate recognition by Escherichia coli
AlkB, a DNA repair enzyme that removes methyl adducts and some
larger alkylation lesions from endocyclic positions on purine and
pyrimidine bases. In vitro Michaelis–Menten analyses on a series of
alkylated bases show high activity in repairing N1-methyladenine
(m1A) and N3-methylcytosine (m3C), comparatively low activity in
repairing 1,N6-ethenoadenine, and no detectable activity in repair-
ing N1-methylguanine or N3-methylthymine. AlkB has a substan-
tially higher kcatand Kmfor m3C compared with m1A. Therefore,
the enzyme maintains similar net activity on the chemically distinct
substrates by increasing the turnover rate of the substrate with
nominally lower affinity. Cocrystal structures provide insight into
the structural basis of this ‘‘kcat/Kmcompensation,’’ which makes a
significant contribution to promiscuous substrate recognition by
AlkB. In analyzing a large ensemble of crystal structures solved in
the course of these studies, we observed 2 discrete global confor-
mations of AlkB differing in the accessibility of a tunnel hypoth-
esized to control diffusion of the O2substrate into the active site.
Steric interactions between a series of protein loops control this
conformational transition and present a plausible mechanism for
preventing O2binding before nucleotide substrate binding.
iron 2-oxoglutarate dioxygenase ? broad substrate specificity ?
ally diverse substrates (1). This phenomenon is sometimes referred
promiscuity,’’ which refers to the ability to perform different types
of chemical reactions. Both phenomena represent important prop-
erties of many naturally evolved enzymes. Promiscuous substrate
recognition is particularly advantageous for DNA-repair enzymes,
because it allows a single enzyme to protect against structurally
diverse types of DNA damage (1). However, the ability of an
enzyme to operate on substrates that differ in size, charge, or
hydrogen-bonding capacity is naively at odds with concepts of
lock-and-key enzymology (2). The ability to bind distinct chemical
structures might also complicate efforts to design specific enzyme
inhibitors. The inhibition of DNA repair enzymes has been advo-
cated as a means to improve cancer therapy, because these enzyme
systems reverse the effects of DNA damaging compounds, which
remain among the most commonly used antitumor agents (3).
Understanding the structural basis for promiscuous substrate rec-
ognition could facilitate inhibitor development, in addition to
providing insight into a fundamentally important enzyme property.
romiscuous substrate recognition refers to the ability of an
enzyme to catalyze equivalent chemical reactions on structur-
A clear example of promiscuous substrate recognition is pro-
vided by AlkB, a direct damage repair enzyme that reverses
chemical lesions generated by SN2 alkylation reagents on the
endocyclic nitrogen atoms of nucleic acid bases (4, 5). This enzyme
has been reported to remove methyl adducts from the N1 atoms of
adenine (m1A) and guanine (m1G) and the N3 atoms of cytosine
(m3C) and thymine (m3T) (4–8), as well as larger ethyl, propyl,
hydroxyalkyl, and exocyclic etheno and ethano adducts from ade-
nine and cytosine bases (Fig. 1) (9–12). Also, AlkB dealkylates
bases in both single- and double-stranded DNA and RNA (4, 5, 13,
in different studies, as well as in the reported specific activities (SI
Appendix, Table S1), it is clear that AlkB recognizes a wide variety
of chemically distinct substrates.
AlkB hydroxylates alkyl lesions on the carbon atom that is
base. This reaction results in an unstable bond that spontaneously
hydrolyzes to restore the unmodified base. The hydroxylation
reaction employs molecular oxygen (O2) as a substrate and 2-oxo-
glutarate (2OG) as a cosubstrate in addition to an Fe(II) cofactor
that catalyzes the redox chemistry. The fold of the catalytic core
in AlkB and the central features of its reaction chemistry are
shared with a superfamily of enzymes called Fe-2OG dioxygenases
(15), which represents the largest known group of nonheme iron
of Escherichia coli AlkB was solved (16) with the enzyme bound to
Fe(II), 2OG, and an m1A containing DNA substrate, i.e., dT-(1-
core of AlkB is homologous to other Fe-2OG dioxygenases (15), a
unique nucleotide-recognition lid is formed by residues from the N
terminus of the enzyme. Dynamic flexibility in this unique subdo-
main was hypothesized to have a role in docking substrates of
varying size into the active site (16).
To elucidate the enzymological and structural basis of promis-
cuous substrate recognition by E. coli AlkB, we present here
coordinated cocrystallization studies and Michaelis–Menten ki-
netic analyses on a series of chemically distinct substrates. In the
course of this study, we observed that the catalytic core of AlkB
undergoes a discrete conformational change (CC) that alters the
relative accessibility of a putative O2-diffusion tunnel leading into
the active site. This CC may prevent premature generation of
Author contributions: B.Y. and J.F.H. designed research; B.Y. performed research; B.Y.
contributed new reagents/analytic tools; B.Y. and J.F.H. analyzed data; and B.Y. and J.F.H.
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
August 25, 2009 ?
vol. 106 ?
no. 34 ?
reactive oxygen intermediates during the multistep redox reaction
cycle of AlkB and other Fe-2OG dioxygenases.
Results and Discussion
Kinetic Analyses of Repair of Model Nucleotide Substrates in Vitro.
We performed Michaelis–Menten analyses on a series of synthetic
trinucleotides with thymine bases flanking a central alkylated base.
Oxidative dealkylation by AlkB-?N11 was quantified using HPLC
and dT-(1,N6-etheno-dA)-dT (T?AT) substrates (10, 11, 16), we
assayed activity on dT-(3-methyl-dC)-dT (TmCT), dT-(1-methyl-
dG)-dT (TmGT), and dT-(3-methyl-dT)-dT (TmTT). Assaying
repair of lesions in a conserved oligonucleotide background en-
abled activity to be compared without interference from variations
in substrate length and sequence identity. To ensure that conclu-
sions did not depend on backbone structure, we also assayed repair
of m1A and m3C in the pentanucleotides dC-dA-(1-methyl-dA)-
dA-dT (CAmAAT) and dC-dA-(3-methyl-dC)-dA-dT (CAm-
CAT). Reaction times were optimized to ensure accurate measure-
ment of initial enzyme velocity without interference from substrate
(SI Appendix, Fig. S1). These assays yielded kcatand Kmvalues for
the TmAT, TmCT, T?AT, CAmAAT, and CAmCAT substrates
(Fig. 2 and Table 1).
are similar to those previously determined for AlkB-?N11 or
full-length AlkB (SI Appendix, Table S1) (10, 16). However, sub-
stantially higher kcat and Km values of 21 min?1and 23 ?M,
respectively, were determined for TmCT. Note that the Km for
TmCT is so high that assays could not be conducted at saturating
substrate concentrations, introducing substantial uncertainty into
is determined accurately even under these circumstances, because
it is given directly by the slope of initial velocity vs. substrate
seen mathematically by differentiation of the Michaelis–Menten
equation). It is noteworthy that the kcat/Kmratio is very similar for
both TmAT and TmCT substrates (1.9 vs. 0.9 min?1?M?1). The
failure to achieve enzyme saturation with TmCT, at concentrations
much higher than those required to achieve saturation with TmAT,
demonstrates that the Kmmust be substantially higher for TmCT.
Futherfore, because kcat/Kmis accurately measured for both sub-
strates, we can conclude that TmCT must also have a higher kcat,
which is also qualitatively clear from the initial-velocity data (Fig.
1). Therefore, the conclusion is robust that both kcatand Kmare
elevated for TmCT, despite uncertainty in the individual values.
CAmCAT (Table 1), where full saturation of the enzyme could be
achieved with both substrates. Irrespective of backbone structure,
the m3C substrate with lower affinity was repaired faster than the
pentamer substrate was very similar to that of the timer containing
the same lesion (Table 1), as expected for identical alkylated bases
if the polynucleotide backbone does not influence active-site ste-
reochemistry. However, the Kmvalues were 20- to 80-fold lower for
the pentamer substrates compared with the trimer substrates
(Table 1), indicating that AlkB bound the longer substrates sub-
observations on some longer DNA substrates (10, 17), higher
affinity for pentamer vs. trimer substrates is consistent with the
observed backbone interactions in the crystal structures of AlkB
cross-linked to double-stranded DNA 13-mers, which show protein
contacts to all of the phosphates corresponding to our pentameric
substrates (but no phosphates further removed from the alkylated
target base) (18).
As noted above, despite the differences in kcatand Kmfor the
m1A vs. m3C lesions, the net catalytic efficiency (the kcat/Kmratio)
is very similar for both lesions when assayed in a consistent
and 97 vs. 78 min?1·?M?1for the pentamers). This conclusion is
(SI Appendix, Table S1) (4, 5, 7, 13, 14, 17). [A discrepancy in our
values for m3C compared with those reported in one study (17) is
discussed in SI Appendix.] Therefore, offsetting changes in kcatand
Kmyield similar net turnover efficiency for the chemically distinct
compensation,’’ makes an important contribution to promiscuous
substrate recognition by AlkB.
Consistent with previous literature (9, 11), the T?AT substrate
substrates. (A) DNA trimers and (B) pentamers were incubated at 37 °C under
standard reaction conditions. AlkB was used at a concentration of 0.1 ?M for
the TmAT (?) and TmCT (‚), 0.002 ?M for CAmAAT (?) and CAmCAT (‚), and
1.0 ?M for T?AT (*). Reactions quenched by addition of EDTA were analyzed
by reversed-phase HPLC, and the dealkylation rate was determined by quan-
tifying substrate and product peak areas in the resulting chromatogram.
Curves represent nonlinear regressions to the noncooperative Michaelis–
of T?AT repair is plotted on the right y axis in A.
Michaelis–Menten analysis of AlkB-?N11 repair activity on model
Fig. 1.Structures of AlkB substrates. Red lesions are repaired by AlkB.
Table 1. Enzyme kinetic parameters for AlkB
2.7 ? 0.8
21 ? 4
0.13 ? 0.05
5.4 ? 1.3
23 ? 10
1.4 ? 0.9
24 ? 5
60 ? 14
0.06 ? 0.01
0.29 ? 0.03
Initial reaction velocities were analyzed by nonlinear regression curve
fitting using Prism software to obtain the kcat, Km, and SE.
www.pnas.org?cgi?doi?10.1073?pnas.0812938106Yu and Hunt
was repaired much less efficiently, with very approximate kcatand
Kmvalues of 0.13 min?1and 60 ?M, respectively (Table 1). After
60 min, only ?20% of T?AT was repaired in a reaction containing
1 ?M enzyme and 10 ?M substrate (SI Appendix and SI Appendix,
Fig. S2.) The kcat/Km ratio (0.002 min?1·?M?1) is 3 orders of
magnitude lower for T?AT than for the equivalent trinucleotide
substrates with m1A or m3C bases, indicating substantially lower
Despite clear separation between methylated and control non-
methylated trimers during HPLC analysis, no detectable repair of
the TmGT or TmTT substrates was observed in our assays using
enzyme concentrations up to 10 ?M in buffers ranging from pH 6
than m1A or m3C (6–9) (SI Appendix, Table S1). However, in
contrast to our results, detectable repair of m1G and m3T has been
observed both in vitro and in vivo (6, 7). Differences in nucleic acid
substrate length (as observed here for trimeric vs. pentameric
substrates and in published work on DNA glycosylases; see ref. 19)
could account for the varying results observed in vitro, whereas
accessory factors could potentially influence recognition of dam-
aged bases in vivo.
Crystal Structures of E. coli AlkB Bound to Different Trinucleotide
Substrates. To characterize the structural mechanisms underlying
promiscuous substrate recognition, E. coli AlkB-?N11 was cocrystal-
lized with TmCT or T?AT substrates. Crystal structures were deter-
mined for AlkB bound to TmCT, 2OG, and either an Fe(II) cofactor
conditions (1.5 Å) (SI Appendix, Tables S2 and S3). There are no
0.29 Å for least-squares superposition of 200 C? atoms. Cocrystalliza-
tion with 12 mM T?AT under aerobic conditions yielded a crystal
site, but no evidence of electron density for the trinucleotide substrate
(SI Appendix, Table S2). To our knowledge, this structure is the first
determined for AlkB bound to a metal cofactor and 2OG without a
structure of the enzyme bound to Fe(II) and succinate, the product of
2OG decarboxylation (16). Also, a higher resolution structure was
determined for AlkB cocrystallized with Fe(II), 2OG, and the TmAT
substrate under anaerobic conditions (SI Appendix, Table S2). This
structure at 1.7-Å resolution is nearly identical to the published 2.3-Å
structure of the same complex (16), giving an rmsd of 0.21 Å for
least-squares superposition of 199 C? atoms. Comparing the higher
resolution Fe-2OG-TmAT structure, which is used in all analyses
presented below, to the equivalent Fe-2OG-TmCT structure shows
much larger conformational differences, giving an rmsd of 0.71 Å for
least-squares superposition of 198 C? atoms. The origin and signifi-
cance of these differences are dissected in detail below.
Two Global AlkB Conformations Differing in Accessibility of the
Putative O2-Diffusion Tunnel. Structural superposition of the com-
plete ensemble of available crystal structures of E. coli AlkB
revealed substantial conformational differences in the double-
stranded ?-helix comprising the catalytic core of this enzyme and
homologous Fe-2OG-dioxygenases. Detailed structural analyses
showed that 64 of 131 residues in the conserved core adopt an
equivalent backbone conformation in all structures, but that most
of the remaining residues adopt 1 of 2 discrete alternative confor-
mations (Fig. 3; SI Appendix, Figs. S3–S5).
When the conserved cores are aligned, concerted conforma-
tional changes (CC1-5) are observed involving 5 different polypep-
tide segments (residues 14–22, 104–111, 136–141, 155–166, and
(Fig. 3). Backbone C? atoms in these polypeptide segments move
3C). Although the loop spanning residues 158–164 (part of CC4)
exhibits the most variable backbone conformation of any segment
involved in the global CC (Fig. 3C; SI Appendix, Fig. S5B), there is
only minimal overlap in the positions it adopts in the 2 conforma-
tions (Fig. 3A; SI Appendix, Fig. S5). Also, the backbone B-factors
in this segment are elevated consistently in only 1 conformation
(blue and green in SI Appendix, Fig. S6). Therefore, the confor-
mation of the 158–164 loop is clearly influenced by the discrete
global CC in AlkB. In contrast, 2 loops in the nucleotide-
recognition lid occupy a continuum of different positions, leading
to their designation as flexible loops (FL1 and FL2 in Fig. 3).
A central feature of the discrete global CC is a coupled rotation
B; SI Appendix, Figs. S3–S5), which are invariant in AlkB orthologs
(16), and make van der Waals contacts to one another in both
conformations. In one conformation, these residues line the wall of
the O2-binding cavity, whereas in the other, they both flip into the
cavity, substantially reducing its volume. When flipped in, the side
and ?-C7, and the resulting movement of this structure pushes the
side chain of L184 into a putative O2-diffusion tunnel that leads
from the surface of the protein to a water-filled O2-binding cavity
adjoining the catalytic iron site. This movement effectively closes
the tunnel (Fig. 3B; SI Appendix, Figs. S3 and S4). Therefore, we
designate the 2 conformations as ‘‘closed’’ (magenta, yellow, and
of the 11 structures that we have determined are in the open
conformation, whereas the remainder are in the closed conforma-
by another group (18).
Allosteric Interactions Coupled to Opening/Closing of the O2-Diffusion
Tunnel. The consistency of the backbone conformation observed in
the open vs. closed structures of AlkB (SI Appendix, Fig. S5)
suggests that the transition between these discrete global confor-
mational states is an intrinsic property of the protein. Also, the fact
that both conformations are observed in crystals with the same
DNA substrate that grew in the same space group from equivalent
solution conditions (Table 2) suggests that this transition involves a
relatively modest free energy change, as observed for functional
allosteric CCs in some other proteins (20). In the crystal lattice, the
transition appears to be controlled by interprotomer packing in-
teractions, because the observed conformational state of the pro-
tein correlates perfectly with the occurrence of alternative packing
contacts near the 158–164 loop (in CC4) and the entrance of the
O2-diffusion tunnel (spanning CC3 and CC5) (SI Appendix, Fig.
could have a functional role in the catalytic reaction cycle of AlkB
changes near the catalytic Fe ion (SI Appendix, Table S4) to CCs in
the 158–164 loop (part of CC5), which interacts weakly with the
trinucleotide in our crystal structures (Fig. 4 and Fig. S8), but more
strongly with the larger nucleotide substrate in the DNA cross-
linked structures (18). This pathway of allosteric communication
could conceivably couple binding of the DNA substrate to changes
in the redox properties of the Fe ion that activate it for catalysis.
DNA binding could also potentially trigger opening of the O2-
diffusion tunnel, which could prevent gratuitous turnover of 2OG
and release of reactive oxygen species before the binding of DNA.
Additional work will be required to critically evaluate these possi-
bilities. More detailed discussions of this material are presented in
The discrete global conformational transition that we have
characterized crystallographically seems to be substantially differ-
ent from the transition recently observed in solution NMR studies
of AlkB bound to 2OG vs. succinate (21). Most of the residues
located in regions of AlkB that do not show significant conforma-
tional differences in the ensemble of available crystal structures (SI
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Appendix, Fig. S9). Residue L184, which gates opening/closing of
ical exchange broadening in the succinate-bound, but not 2OG-
bound conformations of AlkB in solution. However, most other
residues involved in the crystallographically observed transition do
not show any comparable effects, so it is unclear whether the
dynamics of tunnel opening are enhanced on conversion of 2OG to
Stereochemistry of 1-Methyladenosine Vs. 3-Methylcytosine Binding.
The original crystal structures of E. coli AlkB bound to TmAT (16)
showed that the alkylated base is sandwiched between the side
superposition of core ?-strands in the Fe-2OG dioxygenase domain [with 2OG, Fe(II), and TmCT shown from just a single structure]. Discrete local CCs are observed in
5 protein segments (CC1–CC5). FLs (FL1 and FL2), which contact the backbone of the trinucleotide substrate, exhibit elevated backbone B-factors as well as
conformational variability not correlated with the conformational state of the enzyme (16). The water molecule replaced by O2to initiate the oxidation reaction
occupies the sixth site on the octahedrally coordinated Fe ion. AlkB-Fe(II)-2OG-TmCT is shown in orange, AlkB-Mn(II)-2OG-TmCT in yellow, AlkB-Fe(II)-2OG-TmAT (at
channel and O2-binding cavity with structures colored as in A. The molecular surface of AlkB-Fe(II)-2OG-TmAT is shown in gray. (C) Plot of C? displacements between
all nucleotide-bound crystal structures after least-squares superposition of core ?-strands. The legend indicates the color/symbol used for pairwise structural
comparisons. Blue and green are used for comparisons between pairs of structures both in the closed or open conformational state, respectively, whereas red is used
for comparisons between structures in different states. For stereo view of B, see SI Appendix, Fig. S3.
The ensemble of AlkB-?N11 crystal structures reveals a discrete change in global protein conformation. (A) Nucleotide-bound structures after least-squares
Table 2. Summary of E. coli AlkB crystal structures
PDB ID LigandsSpace group ConformationRef.
For expanded version, see Table S3.
†Only crystal form with 2 protomers in the asymmetric unit.
TAGGTAA(m1A)AC*CGT; DNA5, TAGGTAA(m1A)AC*CGT.
www.pnas.org?cgi?doi?10.1073?pnas.0812938106 Yu and Hunt
chains of invariant residues W69 and H131 in a deep binding slot
terminating at the catalytic iron center (Fig. 4). The polynucleotide
backbone is held in place primarily by contacts to side chains on 3
flexible regions in the protein (FL1, FL2, and R161 in CC4). It was
suggested that the flexibility of these loops would enable alkylated
while maintaining equivalent contacts to the invariant polynucle-
otide backbone (i.e., by sliding the base into or out of the slot, as
necessary) (16). The crystal structures of AlkB bound to TmCT
reported in this article confirm this prediction, showing partial
compensation for the change in base dimensions via this mecha-
crystal structure of the human AlkB homolog ABH2 (18), but not
in the nucleotide-free structure of ABH3 (SI Appendix and SI
Appendix, Fig. S10) (22).
Detailed stereochemical analyses must be performed on struc-
tures in the same global conformational state to avoid confusing
effects related to opening the O2-diffusion tunnel with those
involved in accommodating alternative nucleotide substrates.
Therefore, Fig. 4 shows a superposition of the Fe(II)-TmCT and
Co(II)-TmAT structures, both of which are in the closed confor-
mation. These structures contain different metal cofactors, but this
difference in cofactors is not a significant complication, because
detailed comparisons of structures containing the same nucleotide
substrate in the same global conformational state show only min-
imal changes in ligand binding geometry with Fe(II), Co(II), or
Mn(II) cofactors (16). After least-squares alignment of the con-
formationally invariant Fe-2OG core, the ribose ring of the target
nucleotide in the TmCT structure is positioned 0.6 Å closer to the
metal cofactor than in the TmAT structure, even though the
polynucleotide backbone makes nearly identical contacts to the
protein in both cases (Fig. 4). This displacement partially compen-
sates for the smaller size of the modified cytosine base by moving
the target methyl atom closer to its position when attached to a
exhibit much larger movements in crystal structures of AlkB
without a bound nucleotide (Fig. 4A; SI Appendix, Fig. S5A).
Therefore, the flexibility of these protein segments, which undergo
an induce-fit CC on DNA substrate binding, directly contributes to
accommodating structurally diverse substrates.
This displacement only compensates for ?1/3 of the difference
are 1.7 Å apart after backbone alignment of the otherwise equiv-
alent trinucleotide substrates (SI Appendix, Fig. S8A), but the
repositioning mediated by the flexible protein loops moves the
is still a 1.2-Å displacement in their locations after aligning either
the conformationally invariant regions of the core ?-strands (Fig.
4B) or the metal cofactor and its liganding atoms (SI Appendix, Fig.
S11). The direction of this displacement is parallel to the plane of
the alkylated base, which is essentially perpendicular to the vector
from the metal ion to the target methyl group. Because of this
geometrical relationship, the 1.2-Å displacement in the position of
the target methyl group produces a more modest 0.3-Å change in
its distance from the metal ion (Fig. 4B). Therefore, the geometric
architecture of the active site itself contributes to promiscuous
substrate recognition by binding the alkylated base with its plane
oriented approximately perpendicular to the vector from the cat-
alytic Fe ion to the target oxidation site. Larger displacements in
absolute substrate position are thereby converted into smaller
changes in the distance from the catalytic Fe ion to the target alkyl
Structural Basis of kcat-Km Compensation. Despite these 2 comple-
mentary strategies for accommodation of structurally diverse sub-
strates, there are still significant differences in atomic structure in
structure with the smaller TmCT substrate, which displays an
?10-fold faster kcat. In contrast, the atomic packing density will be
higher for the bulkier T?AT substrate (SI Appendix and Fig. S12),
tosine-bound structures. (A) Active site stereochemistry in the Co(II)-2OG-
TmAT (green), Fe(II)-2OG-TmCT (magenta), and Mn(II)-2OG (gray) structures.
Invariant residues H131, D133, and H187 chelate the metal ion cofactor (M),
whereas conserved residues R204 and R210 interact with 2OG. Residue R210
also makes van der Waals contacts to the target methyl group. The partially
disordered phosphates on the 5? terminal thymine are omitted for clarity, as
are water molecules other than that occupying the sixth coordination site on
the metal. (B) Magnified view of the methylated DNA bases in the Co(II)-2OG-
TmAT (green) and Fe(II)-2OG-TmCT (magenta) structures. Nitrogen and oxy-
gen atoms are colored blue and red, respectively, whereas carbon atoms are
colored like the protein backbone. Only the methylated bases and flanking
phosphates of the nucleotide substrates are shown. The purple arrow is
aligned approximately parallel to the 0.6-Å displacement of the ribose group
of the alkylated nucleotide in the m3C vs. m1A structures. This displacement
effectively slides the alkylated base deeper into a binding slot that is approx-
imately coplanar with the sidechain rings of W69 and H131 and terminates at
the catalytic iron center. For stereo views, see SI Appendix, Fig. S8.
Stereochemical comparison of 1-methyadenosine vs. 3-methylcy-
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