Abstract |
Methylation is a process of high importance, acting as a regulatory mechanism for
nucleic acids and proteins, while it can drastically affect the bioactivity of small
molecules. Chemical methylation's toxicity issues and non-selectivity, highlights the
need for alternative methodology, such as the biocatalytic methylation.
Methyltransferases (MTs) are enzymes that are able to methylate O-, N-, S-, C- atoms
under mild conditions and in aqueous media, with high regio- and chemo-selectivity.
In this thesis, isoeugenol-O-ΜΤ from Clarkia breweri (IeOMT) was studied. This MT
was previously engineered to accept caffeic acid, other phenolic compounds and
flavonoids as substrates. The aim of the current work was to further investigate the
applicability of this MT and its variants against more spatial demanding and bulkier
substrates, such as esculetin, ellagic acid and catecholamines, in order to understand
the features that shape the substrate scope of these enzymes. Herein, we screened
the wild-type enzyme and all in-house available variants of IeOMT, constructed
variants in the framework of this thesis, and small libraries prepared with rational and
semi-rational design.
Esculetin was efficiently converted from the wild-type O-MT and all mutants that where
active against caffeic acid; interestingly, the regioselectivity patterns were altered
compared to the one exhibited with caffeic acid. Ellagic acid was not readily accepted
from the available variants. Several rational single mutants were constructed, targeting
the substrate tunnel broadening, but no conversion was observed. These variants were
also used for caffeic acid and esculetin methylation. Interestingly, uncyclized and
cyclized substrates portrayed differences in their behavior as substrates for enzymatic
reactions. The rational engineering efforts for the acceptance of catecholamines were
not successful, despite targeting several positions in the active site.
Lastly, biocatalytic methylation application is limited by the high cost and instability of
the cofactor (S)-adenosyl-L-methionine (SAM). An enzymatic cascade with a halide
methyltransferase was established and optimized for regeneration of SAM using
catalytic amounts of (S)-adenosyl-L-homocysteine and equimolar amounts of MeI,
regarding the substrate. In cascade reactions on targeted substrate esculetin, a
cascade reaction with T133M mutant of O-MT and wild-type of HMT from Arabidopsis
thaliana led to almost full conversion, with approximately 120 cycles of cofactor
regeneration. This grants the process economically viable and holds promise for large
scale industrial applications.
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