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404 18 Methyltransferases in Biocatalysis

prepared and used as cofactors in the MT-catalyzed alkylation of substrates. The
acceptance of these cofactors by many different MTs [43, 46–49] demonstrates a
broader applicability of these artificial cofactors and may lead to new developments
and applications in biocatalysis, molecular biology, synthetic biology, and medicinal
diagnostics and therapy. However, while DNA MTs are often promiscuous in their
acceptance of cofactor analogs, other MTs, for example, some alkaloid N-MTs, are
often very specific and restricted to conversion of the natural cofactor SAM.
Synthetic cofactors and the promiscuity of many DNA MTs for these cofactors
paved the way for the development of techniques such as the sequence-specific
covalent labeling of DNA. With this method, uncertainties regarding label disso-
ciation due to variation of conditions are avoided [50]. Another application of the
sequence-specific MT-induced labeling of DNA (SMILing DNA) is the incorpora-
tion of genomic tags for optical mapping of DNA-binding proteins [51]. A DNA-MT
modifying a rare sequence in the genome has been chosen to design a ‘‘barcode’’
which identifies the orientation and exact identity of the observed DNA. Applying
this method, a fivefold higher precision in the assignment of the binding site was
observed compared to assigning the promoter location based on the DNA ends.
Bacterial MTs have been shown to be capable of adding formaldehyde to cytosine
residues in DNA to yield 5-hydroxymethylcytosine (hmC) (Scheme 18.8a) [52]. The
enzymes are able to bind hmC-containing DNA and also replace the OH group by
sulfur- or selenium-containing moieties (Scheme 18.8b) [53].
In order to increase the efficiency of alkyl transfer from SAM analogs bearing a
propargylic side chain, the cofactor pocket of the DNA-MT M.HhaI was subjected
to enzyme engineering. The steric properties of the active site were changed
by replacement of three nonessential positions. The transalkylation activity was
improved in the double and triple variants because of a slight increase of cofactor
binding affinity and a significant enhancement of the reaction rate. At the same
time, the methylation rate was reduced, thus enabling competitive alkylation in the
presence of SAM. The same concept was applied to M2.Eco31I as well [54].
A propynyl moiety instead of methyl also has been transferred to tRNA and
′
pre-mRNA catalyzed by RNA 2 -O-MTase from Pyrococcus abyssi, a thermophilic
archaeon, using a synthetic cofactor [55]. Terminal alkyne groups can be subjected
to click reaction in order to introduce chromophores or affinity tags.
A selenium-based cofactor analog has been synthesized and used for substrate
labeling with a protein MT. The replacement of sulfur by selenium significantly
increased the stability of the synthetic cofactors, especially for those bearing
terminal alkyne residues. SAH derivatives with terminal alkynes decompose in
aqueous media. One pathway is water addition to the alkyne and subsequent
lactonization under displacement of the sulfide. Selenonium derivatives decompose
more slowly than sulfonium derivates. At the same time, Se leads to a higher
reactivity in the cofactor synthesis. A broad spectrum of protein MTs acting on
lysine, arginine, and glutamine residues was screened and alkylated with the seleno
derivatives [56].
Capture compounds (Figure 18.3) as used in activity-based protein profiling
(ABPP) have been designed for MT profiling based on modified SAH 2 and are