Kim Ahram, Stewart Jon D
Department of Chemistry, University of Florida, 126 Sisler Hall, Gainesville, Florida 32611, United States.
Biochemistry. 2025 Jan 7;64(1):238-249. doi: 10.1021/acs.biochem.4c00599. Epub 2024 Dec 17.
5-Aminolevulinate synthase (ALAS) is a PLP-dependent enzyme that catalyzes the production of 5-aminolevulinate from succinyl-CoA and glycine. Its ability to catalyze the essentially irreversible - bond formation has significant potential in chemoenzymatic synthesis of α-amino ketones. Native ALAS, unfortunately, is extremely substrate-selective, and this seriously limits its synthetic utility. Here, we have used three different protein engineering strategies to overcome this problem for the acyl-CoA substrate. By combining previously reported mutation results and structural analysis, a series of site-saturation mutagenesis/screening efforts were focused on R21, T82, N84, and T362 of ALAS. These yielded single, double, and triple mutants with significantly improved substrate ranges. The steady-state kinetic parameters of several key variants were determined. These data were analyzed in the framework of the ALAS catalytic mechanism to identify the steps that may have been impacted. The most active variant was used in a larger-scale reaction to demonstrate its synthetic potential. Taken together, our results show how ALAS might become a useful biocatalyst for α-amino ketone synthesis and have also allowed us to comment on the relative merits of each the three protein engineering strategies utilized.
5-氨基乙酰丙酸合酶（ALAS）是一种依赖磷酸吡哆醛的酶，它催化琥珀酰辅酶A和甘氨酸生成5-氨基乙酰丙酸。其催化基本不可逆的碳-碳键形成的能力在α-氨基酮的化学酶促合成中具有巨大潜力。不幸的是，天然的ALAS对底物具有极高的选择性，这严重限制了其合成应用。在此，我们采用了三种不同的蛋白质工程策略来克服酰基辅酶A底物的这一问题。通过结合先前报道的突变结果和结构分析，一系列位点饱和诱变/筛选工作聚焦于ALAS的R21、T82、N84和T362位点。这些工作产生了底物范围显著改善的单突变体、双突变体和三突变体。测定了几个关键变体的稳态动力学参数。在ALAS催化机制的框架内对这些数据进行了分析，以确定可能受到影响的步骤。使用活性最高的变体进行了更大规模的反应，以证明其合成潜力。综上所述，我们的结果表明了ALAS如何可能成为α-氨基酮合成的有用生物催化剂，也使我们能够对所采用的三种蛋白质工程策略各自的优点进行评论。
