Abstract
The enzymatic reactions leading to the deamination of β-lysine, lysine, or 2-aminoadipic acid are of great interest for the metabolic conversion of lysine to adipic acid. Enzymes able to carry out these reactions are not known, however ammonia lyases (EC 4.3.1.-) perform deamination on a wide range of substrates. We have studied 3-methylaspartate ammonia lyase (MAL, EC 4.3.1.2) as a potential candidate for protein engineering to enable deamination towards β-lysine, that we have shown to be a competitive inhibitor of MAL. We have characterized MAL activity, binding and inhibition properties on six different compounds that would allow to define the molecular determinants necessary for MAL to deaminate our substrate of interest. Docking calculations showed that β-lysine as well as the other compounds investigated could fit spatially into MAL catalytic pocket, although they probably are weak or very transient binders and we identified molecular determinants involved in the binding of the substrate. The hydrophobic interactions formed by the methyl group of 3-methylaspartic acid, together with the presence of the amino group on carbon 2, play an essential role in the appropriate binding of the substrate. The results showed that β-lysine is able to fit and bind in MAL catalytic pocket and can be potentially converted from inhibitor to substrate of MAL upon enzyme engineering. The characterization of the binding and inhibition properties of the substrates tested here provide the foundation for future and more extensive studies on engineering MAL that could lead to a MAL variant able to catalyse this challenging deamination reaction.
Highlights
In order to move from an oil-based economy to a bio-based economy, reliable processes must be established for the production of a broad range of chemicals important in modern societies
The well-characterized methylaspartate ammonia lyase (MAL) enzymes Citrobacter amalonaticus (CaMAL), CtMAL and ChMAL were selected based on the extensive information available on their catalytic mechanism, their biochemical properties and the availability of their crystal structures [22,23]
Recombinant CaMAL, CtMAL and ChMAL were produced in Escherichia coli and showed activity towards the natural substrate 3-methylaspartate (Table 1)
Summary
In order to move from an oil-based economy to a bio-based economy, reliable processes must be established for the production of a broad range of chemicals important in modern societies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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