E-Locus - Institutional Repository of the University of Crete

Home Collections Type of Work Post-graduate theses

Post-graduate theses

Current Record: 23 of 6511

[Add to Basket]

Identifier

000461547

Title

Investigations on the substrate scope and subsequent evolution of O-Methyl Transferases for the establishment of methylation bioprocesses

Alternative Title

Μελέτη του φάσματος υποστρωμάτων και επακόλουθη εξέλιξη Ο-Μεθυλοτρανσφερασών για την ανάπτυξη βιοδιεργασιών μεθυλίωσης

Author

Χατζάκης, Εμμανουήλ Α.

Thesis advisor

Παυλίδης, Ιωάννης

Reviewer

Γανωτάκης, Δημήτριος
Ελευθεριάδης, Νικόλαος

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.

Language

English

Subject

Esculetin

O-MTS

Substrate scope

Εσκουλετίνη

Μεθυλοτρανσφεράσες

Φάσμα υποστρωμάτων

Issue date

2024-03-21

Collection