Catalytic Space Engineering as a Strategy to Activate C-H Oxidation on 5-Methylcytosine in Mammalian Genome

Article abstract of DOI:10.1021/jacs.1c03815

Conditional remodeling of enzyme catalysis is a formidable challenge in protein engineering. Herein, we have undertaken a unique active site engineering tactic to command catalytic outcomes. With ten-eleven translocation (TET) enzyme as a paradigm, we show that variants with an expanded active site significantly enhance multistep C-H oxidation in 5-methylcytosine (5mC), whereas a crowded cavity leads to a single-step catalytic apparatus. We further identify an evolutionarily conserved residue in the TET family with a remarkable catalysis-directing ability. The activating variant demonstrated its prowess to oxidize 5mC in chromosomal DNA for potentiating expression of genes including tumor suppressors.

Full text of DOI:10.1021/jacs.1c03815

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Catalytic Space Engineering as a Strategy to Activate C−H Oxidation
on 5‑Methylcytosine in Mammalian Genome
Sushma Sappa, Debasis Dey, Babu Sudhamalla, and Kabirul Islam*
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ABSTRACT: Conditional remodeling of enzyme catalysis is a formidable challenge in protein engineering. Herein, we have
undertaken a unique active site engineering tactic to command catalytic outcomes. With ten−eleven translocation (TET) enzyme as
a paradigm, we show that variants with an expanded active site significantly enhance multistep C−H oxidation in 5-methylcytosine
(5mC), whereas a crowded cavity leads to a single-step catalytic apparatus. We further identify an evolutionarily conserved residue in
the TET family with a remarkable catalysis-directing ability. The activating variant demonstrated its prowess to oxidize 5mC in
chromosomal DNA for potentiating expression of genes including tumor suppressors.
nzymes that modify postreplicative genetic material are
crucial for safeguarding and accessing the information
E
encoded therein. DNA methyltransferase, which methylates
carbon 5 on cytosine to form 5-methylcytosine (5mC) to drive
mammalian gene expression, is a prime example.1 5mC has
been shown to undergo iterative C−H oxidation by the ten−
eleven translocation (TET) enzymes to 5-hydroxymethylcyto-
sine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine
(5caC) in the presence of 2-ketoglutarate (2KG). Subse-
quently, 5fC and 5caC are both excised by thymine DNA
glycosylase (TDG) for repair, constituting an intriguing
mechanism for DNA demethylation (Figure 1A).2−6 Due to
their distinct stereoelectronic features, the 5mC oxidative
intermediates (5mCox) interact with a range of chromatin-
associated proteins and regulate gene expression.7−9
Much is unknown about the context-dependent functions of
the intermediates as demethylation mediators and transcrip-
tional regulators. Assigning a distinct and compensatory role to
5mCox species, particularly 5fC and 5caC, in mammalian
physiology has remained a challenge due to their intrinsic low
abundance and fleeting existence. Limited efforts to modulate
5mCox distribution in DNA include ascorbic acid mediated
TET activation.10 Ascorbic acid, however, activates several
2KG-dependent dioxygenases, obscuring its exclusive role in
controlling TET activity. Other approaches focus on condi-
tional reconstitution of wild-type TET2, which are less likely to
regulate the 5mCox distribution.11,12
We envisioned an active site remodeling tactic to stimulate
C−H oxidation for enriching the human genome with the low-
abundant intermediates for functional analysis. In TET-
mediated C−H oxidation, hydrogen abstraction by oxo-ferryl
species is the rate-limiting step with a relative activation barrier
following the order 5fC > 5hmC > 5mC (Figure 1B).13
Computational analyses indicate that restrained conformations
of 5hmC and 5fC within the active site likely prevent the
abstractable hydrogens from adopting a favorable orientation
for C−H oxidation; in contrast, all the rotational conformers of
5mC lead to productive hydrogen abstraction.14−16 We
Figure 1. Catalytic cycle of TET-mediated 5mC oxidation. (A)
Cytidine demethylation by TET and TDG. (B) In a catalytic cycle,
proton abstraction by FeIV(O) constitutes the rate-determining
transition state. (C) Engineering active site space of TET enzymes
to control the 5mCox distribution in DNA.
Received: April 11, 2021
https://doi.org/10.1021/jacs.1c03815
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© XXXX American Chemical Society
A

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reasoned that systematic alteration of the stereoelectronic
component at the TET−2KG−5mC interface could modulate
the transition state barrier toward a more controlled product
distribution (Figure 1C).
To test our hypothesis, we generated a panel of TET2
mutants by replacing several hydrophobic and polar residues in
the active site with alanine, one at a time (Figure 2A).17
most notable difference was observed for the V1395A mutant,
as it generated 5caC as the dominant product (>90%) (Figure
2C,D). To the best of our knowledge, such an activating TET
mutant is yet to be reported. Collectively, our systematic
screening experiment identified TET2 variants that could
significantly modulate 5mCox distribution by either stalling the
oxidation pathway at the first step to exclusively generate
5hmC or accelerating to the fully oxidized product. The
V1395A mutant is particularly important given that 5caC is
rare in the mammalian genome.
We further analyzed 5caC formation using base-resolution
sequencing. It has been shown that 5caC is deaminated upon
treatment with bisulfite and read as T during sequencing
(Figure 3A).21 In contrast, 5mC and 5hmC remain as C due to
Figure 2. Active site engineering toward novel TET2 variants. (A)
Structure of TET2 bound with 5mC DNA (PDB: 4NM6).17 (B)
Percent intensities of MALDI-MS signals for a range of mixtures of
5mC DNA with either 5hmC, 5fC, or 5caC. (C) Heat-map
representation of % 5mCox furnished by TET2 mutants. (D)
MALDI-MS spectrum showing activating effect of V1395A to
generate 5caC.
Figure 3. Analysis of 5mCox using Sanger sequencing. (A) Scheme
showing bisulfite-mediated 5caC deamination and decarboxylation.
(B) V1395A-catalyzed 5mC oxidation followed by bisulfite treatment
and PCR, confirming 5caC as the major product based on Sanger
sequencing data. BS = bisulfite.
Employing a MALDI-MS-based assay, we examined the
oxygenase activity of these mutants toward a short dsDNA
substrate, CAC5mCGGTG.18,19 To analyze the product
distribution in a quantitative manner, we first examined if
the DNA segments carrying various 5mCox intermediates
displayed comparable ionization potentials. For this, we
synthesized three additional DNAs (CACXGGTG; X =
5hmC, 5fC, and 5caC) (Figure S1, Schemes S1−3). 5mC
DNA was mixed with each synthesized 5mCox DNA separately
in different ratios and subjected to MALDI-MS. Peak intensity
of the individual DNA reflected their known ratio in the
mixture, indicating equivalent degrees of ionization in MALDI
analyses (Figures 2B, S2−S4).
their inertness toward the reagent. We subjected a duplex 76-
mer DNA carrying a central 5mCpG unit to TET-mediated
oxidation followed by bisulfite treatment, PCR amplification,
and Sanger sequencing. While 5mC in samples exposed to
either no protein or wild-type TET2 read as C, the equivalent
site in the V1395A-treated sample emerged as T (Figures 3B,
S6). These results demonstrate the ability of the mutant to
predominantly generate 5caC.
In an in vitro assay, wild-type TET2 converted 5mC DNA to
5hmC, 5fC, and 5caC. In contrast, the mutants oxidized
substrate DNA with varied degrees of specificity (Figures 2C,
S5). T1372A, T1393A, and T1883A mutants showed
remarkable specificity for generating primarily 5hmC. It has
been shown that a H-bonding network involving T1372 is
critical for rotating 5hmC to switch from product to substrate
orientation for further oxidation; lack of such interaction in
T1372A locks 5hmC in the product conformation.20 We
suspect a similar mechanism is operating for T1393A and
T1883A mutants as well to impart the degree specificity in C−
H oxidation. Among the hydrophobic residues screened, the
We next compared the activity of V1395A with that of wild-
type TET2 on three individual substrates (Figures 4A−C, S7−
S12). With respect to the wild-type enzyme, V1395A led to a
significantly higher production of 5hmC from 5mC, while both
enzymes showed comparable activity on 5hmC DNA. The
mutant displayed further improved activity on 5fC leading to
higher 5caC formation, demonstrating that it is indeed acting
as a superior dioxygenase in two discrete cycles involving
conversions of 5mC to 5hmC and 5fC to 5caC in a three-step
catalytic process. To further gauge the relative activity, we
measured catalytic efficiencies of V1395A and wild-type TET2
on 5mC with varying concentrations of 2KG (Figure S13).
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Such mutants are valuable for probing member-specific activity
of TET1−3 with identical catalytic pockets but distinct
expression patterns and domain organizations.27
We next examined the ability of the V1395A mutant to
oxidize 5mC on genomic DNA isolated from HEK293T cells.
The product distribution was analyzed in a dot-blot assay using
5mCox-specific antibodies. The mutant furnished 5fC and 5caC
more than 3-fold in excess compared to wild type, a result
consistent with the variant having a higher enzymatic activity
(Figures 5A, S19). It is notable that the variant with an
Figure 4. Characterization of the TET variants. (A−C) Time-
dependent activity of wild-type TET2 and V1395A toward 5mC (A),
5hmC (B), and 5fC (C) substrate DNAs. (D) Heat-map
representation of % 5mCox generated by V1395A congeners. (E)
Sequence alignments of eukaryotic TETs highlighting the gatekeeper
residue. (F) Activity of wild-type TET1 and the V1685A mutant on
substrate DNAs.
While the mutant had improved turnover (kcat), both enzymes
showed comparable KM values, ruling out the possibility that
enhanced activity of V1395A could be from improved cofactor
binding.
Figure 5. Activity of TET2 and V1395A on genomic DNA. (A) Dot-
blot assay showing time-dependent increase in 5caC by V1395A. The
bar diagram provides relative fold changes in 5caC. (B) Chemical
strategy to selectively derivatize 5caC. (C) Dot-blot assay with 5aC
and biotin antibodies before and after derivatization, respectively. Bar
representation of biotinylated 5caC on genomic DNA. Nonlinearity in
biotin signal is likely due to multistep functionalization of 5caC.
To further examine the influence of active site space on
product distribution, we generated a panel of V1395 mutants
differing in amino acid side chains (Figures 4D, S14). As
expected, the glycine variant generated 5caC as the major
product. Remarkably, the mutants with a crowded active site
furnished primarily 5hmC, demonstrating a clear relationship
between catalytic pocket size and 5mCox distribution. This
switch from three-step catalysis (5mC → 5hmC → 5fC →
5caC) by V1395A to just one (5mC → 5hmC) by its
congeners thus must be a result of crowding the active site.
Intrigued by the unique catalysis-directing feature of V1395
in TET2, we analyzed the sequences of representative TET
homologues and found that a bulky hydrophobic residue at
equivalent sites is strictly conserved among eukaryotes,
indicating its gatekeeper-like role in controlling 5mCox
derivatives (Figure 4E).22−26 We further noted that the
space-creating mutation is absent in human genome variants
(Figure S15, Table S1). To access a similar activating variant of
human TET1, we generated a V1685A mutation that indeed
showed remarkably higher enzymatic activity compared to
wild-type TET1 (Figures 4F, S16−S18). Our work thus
identifies a conserved gatekeeper residue in eukaryotic TETs
that can be engineered to regulate 5mCox content in DNA.
expanded active site is capable of generating an excess of 5caC
on the entire human DNA carrying millions of 5mC moieties.
Remarkably, V1685A also enhanced 5mCox content on
genomic DNA in comparison to wild-type TET1, consistent
with the activating nature of the expanded catalytic site (Figure
S20).
This result prompted us to apply the TET2 variant in
combination with a chemo-functionalization strategy to enrich
5caC-containing DNA (Figure 5B).21,28 To selectively
derivatize 5caC, the 5fC group in TET-treated DNA was
reduced using NaBH4 followed by coupling with biotinylated
hydroxylamine on an activated carboxylate substrate. Sub-
sequently, the samples were analyzed in a dot-blot assay using
5caC- and biotin-specific antibodies to gauge pre- and
postfunctionalized 5caC (Figure 5C). Consistent with the
mutant’s enhanced prowess to generate 5caC, we observed a
higher signal of biotinylated 5caC for the mutant over wild-
type TET2, implying that the activating variant can be
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exploited for improved isolation of the low-abundant
intermediates.
To examine if the TET2 variant could activate 5mC
oxidation on the DNA within human cells, we expressed the
catalytic domain of wild-type TET2 or the mutant in
HEK293T cells (Figure 6A). Dot-blot analysis of the isolated
are known to downregulate tumor suppressors in hemato-
logical malignancies.31 We focused on DACT1, a tumor
suppressor that is frequently silenced in cancers via promoter
methylation.32 It has been shown that the transcription factor
WT1 interacts with TET2 to regulate expression of DACT1 in
normal hematopoietic stem cells via 5mC oxidation (Figure
6E).33 In Western blot, we noted that wild-type TET2
marginally increased the DACT1 level in cells compared to a
control vector. Remarkably, the mutant catalyzed DACT1
expression up to 2-fold increase over TET2, consistent with its
higher dioxygenase activity toward 5mC (Figures 6F, S22).
Such results imply that the activating mutant is capable of
enhancing the expression of a tumor suppressor that is
aberrantly silenced in cancers.
In this work, we explored active site space engineering as a
novel tactic to direct catalytic outcome. Mutations at an
evolutionarily conserved residue led to TET variants with
tunable oxygenase activity: an enlarged cavity augmented
multistep oxidation of 5mC to 5caC as the dominant product,
whereas reduced catalytic space led to primarily 5hmC via a
single-step oxidation. Remarkably, the spacious variant
exhibited superior activity to potentiate expression of
antimetastatic genes in human cells. We anticipate the
engineered TET in combination with genome editing
strategies would constitute a powerful tool for establishing a
locus-specific 5mCox pattern to reprogram downstream
processes. The V1395A mutant will find further application
in the enrichment and analysis of 5caC content in the genome
using TET-assisted bisulfite34 and related sequencing ap-
proaches. Furthermore, our work provides a blueprint for
remodeling the catalytic apparatus of 2KG-dependent
dioxygenases, which are emerging as critical regulators of
mammalian gene expression.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c03815.
■
Figure 6. In cellulo activity of TET2 variant. (A) Schematic displaying
activity of V1395A on chromosomal DNA. (B) Dot-blots confirming
higher level of 5hmC and 5caC generated by the mutant. (C) Steps
involved in DIP-qPCR. (D) Fold change (mutant/wild-type) in
enrichment of selected genes using 5caC antibody. (E) TET2
interacts with WT1 to regulate DACT1. (F) Expression of DACT1 is
enhanced by V1395A. MB = methylene blue.
sı
Methods for phosphoramidite synthesis, protein ex-
pression, biochemical assays, and supplementary figures
and tables (PDF)
Table S1: TET1−3 exome summary analysis (XLS)
DNA revealed the presence of ∼2-fold higher 5hmC and 5caC
DNA in V1395A-expressing cells compared to those expressing
the wild-type enzyme (Figures 6B, S21), demonstrating that
the activating effect of V1395A is well preserved in the
complex environment of mammalian cells.
AUTHOR INFORMATION
Corresponding Author
■
Kabirul Islam − Department of Chemistry, University of
Pittsburgh, Pittsburgh, Pennsylvania 15260, United States;
orcid.org/0000-0002-8680-6130; Email: kai27@pitt.edu
We then assessed the ability of V1395A to oxidize 5mC at
individual promoters. We selected a panel of eight genes
including microRNA-200 and EPCAM, as TET-mediated
oxidation of promoter 5mC leads to reactivation of the
corresponding transcripts.29 We performed DNA immunopre-
cipitation (DIP) from HEK293T cells expressing wild-type or
the variant enzymes with 5mCox-specific antibodies followed
by quantitative polymerase chain reaction (qPCR) using gene-
specific primers (Figure 6C).30 Results from independent
replicates suggested that the mutant indeed boosted 5mC
oxidation at these loci compared to wild-type TET2 as
revealed by ∼2-fold enrichment of the genes in the DIP-qPCR
assay (Figures 6D, S21).
Authors
Sushma Sappa − Department of Chemistry, University of
Pittsburgh, Pittsburgh, Pennsylvania 15260, United States
Debasis Dey − Department of Chemistry, University of
Pittsburgh, Pittsburgh, Pennsylvania 15260, United States;
orcid.org/0000-0002-3612-860X
Babu Sudhamalla − Department of Chemistry, University of
Pittsburgh, Pittsburgh, Pennsylvania 15260, United States;
Present Address: Department of Biological Sciences,
Indian Institute of Science Education and Research,
Kolkata, West Bengal, India 741246
Finally, we examined if V1395A is functionally relevant to
potentiate the expression of tumor suppressors. This is
particularly important given that inactivating TET2 mutations
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c03815
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Changes, Electric Field, and Mutations. ACS Catal. 2021, 11 (7),
3877−3890.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the University of Pittsburgh, the National Science
Foundation (MCB-1817692), and the National Institutes of
Health (R01GM123234, R01GM130752) for financial support
and Dr. D. Chakraborty and members of our laboratory for
editing of the manuscript.
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