Biennial Report 2011–2012

Engineering the catalytic reaction
of methyltransferases for targeted
covalent labeling of DNA
G. Lukinavičius, V. Masevičius,
G. Urbanavičiūtė, R. Gerasimaitė, M. Tomkuvienė
We had synthesized a series of model AdoMet analogs with
sulfonium-bound extended side chains replacing the methyl
group and showed that allylic and propargylic side chains
can be efficiently transferred by DNA MTases with high sequence-
and base-specificity (Dalhoff et al, Nature Chem.
Biol., 2006, 2: 31–32; Klimašauskas and Weinhold, Trends
Biotechnol., 2007, 25: 99–104) in collaboration with the
group of Prof. Elmar Weinhold (RWTH Aachen, Germany).
Using DNA MTases along with their novel cofactors that carry
useful functional or reporter groups, we demonstrated that
our new approach name mTAG (methyltransferase-directed
Transfer of Activated Groups) can be used for sequence-specific
functionalization and labeling of a wide variety of model
and natural DNA substrates (Lukinavicius et al., J. Amer.
Chem. Soc., 2007, 129: 2758).
Figure 3. Sequence-specific covalent modification of DNA (N and X =
nucleotide pairs, XXXXX = recognition sequence of the MTase, X = target
nucleotide) catalyzed by a large number of DNA methyltransferases
(MTase). Left, methyltransferase-directed Transfer of Activated Groups
(mTAG) carrying a reactive functionality (green sphere) from a doubleactivated
AdoMet analog onto a target nucleobase in DNA. Right,
Model of the active site of the WT M.HhaI MTase highlights residues
Q82, Y254S and N304 whose sidechains occur in close proximity to the
extended transferrable group of the bound cofactor analog.
HPLC-MS analysis performed by senior scientist Dr. V.Masevičius
Although useful in certain applications, our previously synthesized
AdoMet analogs carrying sulfonium-bound 4-substituted
but-2-ynyl side chains exhibited short lifetimes in
physiological buffers. Examination of the reaction kinetics
and products showed a fast pH-dependent addition of a
water molecule to their transferrable groups. This side reaction
was eradicated by synthesis of a new series of analogs, in
which the separation between an electronegative group and
the triple bond was increased from one to three carbon units.
The designed hex-2-ynyl moiety-based cofactor analogs (Fig.
3, left) with terminal amino, azide or alkyne groups showed
a markedly improved enzymatic transalkylation activity and
proved highly useful for two-step sequence-specific labeling
of DNA using engineered DNA C5-MTases [7].
To further optimize the efficacy of the mTAG reactions we
performed steric engineering of the cofactor pocket in the
M.HhaI C5-MTase by systematic replacement of three nonessential
positions, located in two conserved sequence motifs
and in a variable region, with smaller residues (Fig. 3, right).
We found that double and triple replacements lead to a substantial
improvement of the transalkylation activity, permitting
competitive mTAG labeling in the presence of AdoMet
in vitro and in cell lyzates. Analogous replacements of two
conserved residues in M.HpaII and M2.Eco31I C5-MTases
also resulted in improved transalkylation activity attesting
a general applicability of the homology-guided engineering
to the C5-MTase family and expanding the repertoire of sequence-specific
tools for covalent in vitro and ex vivo labeling
of DNA [3].
Vilnius University Institute of Biotechnology Biennial report for 2011–2012 37