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 ?
which displays an ?20-fold slower kcat (Table 1). Given this
correlation, the faster kcatobserved for the m3C lesion could be
attributable to its lower packing density in the active site. Alterna-
tively, it could be attributable to the 1.2-Å shift in the location of its
methyl group compared with that of m1A. One or both of these
for m3C vs. m1A, due to either quantum-mechanical effects
influencing the rate of electronic rearrangements, or steric effects
influencing the rate of movement of reactive atoms or product
molecules during the multistep redox reaction.
(Fig. 1), its reduced affinity (i.e., higher Km) is likely to be
attributable to having less hydrophobic surface area buried in the
substrate binding slot. The active site stereochemistry in AlkB
seems likely to have evolved to maximize the turnover rate for the
smaller m3C substrate to compensate for its lower binding affinity
2 predominant physiological substrates (23). These compensating
changes in kcatand Kmmake an important contribution to promis-
cuous substrate recognition by AlkB.
Conclusions. The redox reaction cycle of AlkB involves complex
local dynamics in the active site, including the diffusional release of
CO2after the initial oxidation of 2OG to succinate, followed by
physical migration of the reactive oxygen atom on the resulting
‘‘oxyferryl’’ [Fe(IV) ? O] intermediate (16). Even though precise
positioning of substrates is required for efficient catalysis via this
multistep redox reaction, AlkB is able to repair substrates of
different sizes and chemical structures. The structural adaptations
described in this study contribute to promiscuous substrate repair
by enabling the enzyme to maintain efficient catalytic geometry for
chemically diverse substrates.
enzymes (1). However, stereochemical constraints combined with
physiological requirements will ultimately impose limits on enzyme
performance. Exposure to DNA-alkylating reagents produces over
a dozen toxic DNA modifications (23). Although N1-adenine and
N3-cytosine account for ?30% of observed lesions (23), the
for the ?10,000-fold lower efficiency of E. coli AlkB in repairing
m1G and m3T compared with m1A and m3C as observed in our
the side-chain carboxylate of D135) (16), which probably enhance
affinity for these substrates while reducing affinity for guanine/
thymine because they have an exocyclic carbonyl group at the
equivalent position (Fig. 1). While AlkB has evolved to promiscu-
ously repair alkylation damage to adenine and cytosine, its 2 most
prevalent physiological substrates, it appears to have optimized its
affinity for these substrates by diminishing its capacity to repair the
comparatively rare guanine and thymine substrates with equivalent
Materials and Methods
Materials. Trinucleotide substrates (TmAT, TmCT, TmGT, and T?AT) with a
by Biosynthesis, Inc. The TmTT, CAmAAT and CAmCAT nucleotide substrates
were synthesized by Midland Certified Reagent Company.
Protein Purification, Crystallization, and X-Ray Structure Determination. Meth-
ods were equivalent to those used for the initial structure determination of
AlkB-?N11 (16). Briefly, protein expressed from a pET plasmid in E. coli
BL21?(DE3) was purified by Ni-NTA chromatography, repassage through a sec-
ond Ni-NTA column after cleavage of the C-terminal hexa-histidine tag by TEV
protease, and gel filtration. Substrates and metals were added to the protein
immediately before crystallization in an anaerobic glovebox (COY Laboratories
PEG 3350, 0.2 M sodium formate. Crystals were passed briefly through 22% PEG
3350, 0.2 M sodium formate, 10% glycerol before freezing in liquid propane for
see SI Appendix. The conformationally invariant segments of the core ?-strands
used for final least-squares alignment comprised residues 19–22, 115–134, 143–
158, 166–178, 186–189, and 204–210.
Enzyme Kinetics. AlkB-?N11 (typically 0.1 ?M) was mixed with the nucleotide
substrate (1–25 ?M) in 50 ?g/mL BSA, 75 ?M Fe(NH4)2(SO4)2, 1 mM 2OG, 2 mM
2 min and quenched with 10 mM EDTA. Relative substrate and product concen-
reversed-phase HPLC analyses on a Phenomenex Luna C18 column (16).
ACKNOWLEDGMENTS. We thank M. Arbing, J. Benach, B. Gibney, G. Verdon,
J. Schwanof, and R. Abramowitz for advice and technical support. This work
was supported by a National Institutes of Health (NIH) R01 grant and an
American Heart Association Established Investigator award (J.F.H.), as well as
by an NIH Protein Structure Initiative grant to the Northeastern Structural
Genomics Consortium. B.Y. is a Research Fellow of the Terry Fox Foundation
of a Canadian Institute of Health Research Fellowship.
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www.pnas.org?cgi?doi?10.1073?pnas.0812938106 Yu and Hunt