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Identifier

000446595

Title

Engineering an (R)-selective amine transaminase for the acceptance of bulky substrates

Alternative Title

Δημιουργία μιας (R)-εκλεκτικής τρανσαμινάσης αμινών για την μετατροπή ογκώδων υποστρωμάτων

Author

Κόνια, Ελένη Ι.

Thesis advisor

Παυλίδης, Ιωάννης

Reviewer

Τσιώτης, Γεώργιος
Mutti, Francesco

Abstract

Amine transaminases catalyze the transfer of an amino group from a primary amine (amino donor) to the carbonyl carbon of an α-keto acid, a ketone or an aldehyde (amine acceptor). The process of amine transfer is called transamination. The reaction is mediated by an organic cofactor, pyridoxal 5’-phosphate (PLP). Because of the industrial potential of ATAs for production of chiral amines, many research efforts have been devoted to improve the catalytic activity of the ATAs. Optically pure amines are valuable products or intermediate compounds for the synthesis of pharmaceuticals and agrochemicals. While amine transaminases are highly enantioselective catalysts, they only accept methyl substituted phenylamines due to the architecture of their active site. This thesis focuses on engineering the substrate scope of an (R)-selective amine transaminase form Luminiphilus syltensis (LS_ATA) towards alkyl substituted amines. A structure-based rational design strategy was implemented starting from a high-resolution crystallographic structure of the LS_ATA that was resolved by our collaborators. We prepared in silico, through MD simulations, the quinonoid intermediate of (R) 1 phenylethylamine, which is a benchmark substrate of amine transaminases. The model of the intermediate helped us gain insight on important structural features in the enzymes active and binding site. Through bioinformatic analysis and visual inspection we identified three residues in the enzyme’s small binding pocket that possibly hinder the acceptance of bulkier substrates. Site directed mutagenesis confirmed that the mutation V37A in the small binding pocket generates a variant with expanded substrate scope toward bulkier compounds, without any loss in enantioselectivity and enzyme stability. The resulting variant successfully deaminated (R) 1-phenylpropylamine and (R) 1 phenylbutylamine, however no asymmetric synthesis of these amines was observed. Mutation in positions S248A and T249A led to deactivation of the enzymes, mainly owe to unreactive complexes between the enzyme and the PLP cofactor.

Language

English

Subject

Biocatalysis

Luminiphilus syltensis

Rational design

Site directed mutagenesis

Βιοκατάλυση

Πρωτεϊνική μηχανική

Τοποειδική μεταλλαξηγένηση

Issue date

2022-03-28