Division of Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
Computational Biology Laboratory, Danish Cancer Society Research Center, Copenhagen, Denmark.
PLoS One. 2020 May 21;15(5):e0233467. doi: 10.1371/journal.pone.0233467. eCollection 2020.
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.
导致β-赖氨酸、赖氨酸或 2-氨基己二酸脱氨的酶促反应对于赖氨酸向己二酸的代谢转化非常重要。然而，能够进行这些反应的酶尚不清楚，而氨裂解酶（EC 4.3.1.-）对广泛的底物进行脱氨。我们研究了 3-甲基天冬氨酸氨裂解酶（MAL，EC 4.3.1.2）作为蛋白质工程的潜在候选物，以使 MAL 能够对β-赖氨酸进行脱氨，我们已经证明β-赖氨酸是 MAL 的竞争性抑制剂。我们已经对 MAL 在六种不同化合物上的活性、结合和抑制特性进行了表征，这些特性可以确定 MAL 脱氨我们感兴趣的底物所需的分子决定因素。对接计算表明，β-赖氨酸以及研究的其他化合物在空间上都可以适应 MAL 催化口袋，尽管它们可能是弱的或非常短暂的结合物，并且我们确定了与底物结合相关的分子决定因素。3-甲基天冬氨酸的甲基形成的疏水性相互作用，以及碳 2 上氨基的存在，在底物的适当结合中起着至关重要的作用。结果表明，β-赖氨酸能够适应并结合 MAL 催化口袋，并且通过酶工程可以潜在地从抑制剂转化为 MAL 的底物。这里测试的底物的结合和抑制特性的表征为进一步和更广泛的 MAL 工程研究提供了基础，这可能导致能够催化这种具有挑战性的脱氨反应的 MAL 变体。
