DEAMINATION REACTIONS AS PART OF THE METABOLIC PATHWAY FOR THE PRODUCTION OF ADIPIC ACID
Poster (konferens), 2018

Deamination of lysine, β-lysine or 2-aminoadipic acid are enzymatic reactions that have great biotechnological interest as part of the metabolic pathways for the production of adipic acid. The enzymatic activity necessary to catalyze the mentioned deamination reactions is defined as ammonia lyase (EC 4.3.1.-) and cleaves the bond between the amine group and adjacent carbon. Whereas ammonia lyases able to act on the target substrates lysine, β-lysine or 2-aminoadipic acid have not been identified so far, ammonia lyases able to act on other amino acids have long been known. We selected 3-methylaspartate-ammonia lyase (MAL, EC 4.3.1.2) as enzyme that potentially could be made active on the target substrates. Three MAL were recombinantly expressed and purified and the activity tested towards the substrates, but no activity was observed. Different MAL single mutant variants potentially able to catalyze the desired reactions were designed using a computational approach, but unfortunately, no activity towards the target substrates was detected. In order to better understand the substrate scope and catalytic mechanism of MAL, different compunds with similar structure to the natural substrate were tried as substrates. MAL showed activity towards aspartic acid, but no towards the other substrates indicating the narrow specificity of the enzyme. Inhibition studies showed that β-lysine was a competitive inhibitor suggesting that the amino group of the substrate need to be in the β-carbon for the binding. 2-aminoadipic acid was shown to be a non-competitive inhibitor. Finally, docking experiments were carried out to understand if the target substrates fit in the catalytic pocket. The study provides a deeper knowledge of the substrate scope and inhibitors of MAL and analyze if and how the target substrates could be deaminated by MAL. Moreover, the study establishes reliable methods for the detection of deamination activity of lysine, β-lysine and 2-aminoadipic acid.

Författare

Matteo Lambrughi

Danish Cancer Research Society Center

Veronica Saez Jimenez

Chalmers, Biologi och bioteknik, Industriell bioteknik

Željka Sanader Maršić

Danish Cancer Research Society Center

Elena Papaleo

Danish Cancer Research Society Center

Lisbeth Olsson

Chalmers, Biologi och bioteknik, Industriell bioteknik

Valeria Mapelli

Chalmers, Biologi och bioteknik, Industriell bioteknik

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Hållbar utveckling

Ämneskategorier

Biokemi och molekylärbiologi

Biokatalys och enzymteknik

Organisk kemi

Styrkeområden

Livsvetenskaper och teknik

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Skapat

2019-01-16