Longwitz Lars, Kamer Marijn D, Brouwer Bart, Thunnissen Andy-Mark W H, Roelfes Gerard
Stratingh Institute for Chemistry, University of Groningen, Groningen 9747 AG, The Netherlands.
Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen 9747 AG, The Netherlands.
ACS Catal. 2024 Dec 3;14(24):18469-18476. doi: 10.1021/acscatal.4c06052. eCollection 2024 Dec 20.
Genetically encoded noncanonical amino acids can introduce new-to-nature activation modes into enzymes. While these amino acids can act as catalysts on their own due to their inherent chemical properties, interactions with adjacent residues in an enzyme, such as those present in natural catalytic dyads or triads, unlock a higher potential for designer enzymes. We incorporated a boron-containing amino acid into the protein scaffold RamR to create an active enzyme for the kinetic resolution of α-hydroxythioesters. We found that a closely positioned lysine residue is crucial for the catalytic activity of the designer enzyme by forming a hybrid catalytic dyad with the boronic acid residue. The enzyme is capable of resolving differently substituted α-hydroxythioesters with good selectivities. High-resolution mass spectrometry, B NMR spectroscopy, and crystal structure analysis of the designer enzyme gave insight into the three steps of the mechanism (substrate binding, hydroxide transfer, product release). Mutations of a residue around the catalytic dyad led to a variant of the enzyme with 2-fold improvement of catalytic activity and selectivity.
基因编码的非标准氨基酸能够为酶引入全新的天然激活模式。虽然这些氨基酸因其固有的化学性质自身就能充当催化剂，但与酶中相邻残基的相互作用，比如天然催化二元组或三元组中的那些残基，能为设计酶开启更高的潜力。我们将一种含硼氨基酸整合到蛋白质支架RamR中，以创建一种用于α-羟基硫酯动力学拆分的活性酶。我们发现，一个位置紧邻的赖氨酸残基通过与硼酸残基形成混合催化二元组，对设计酶的催化活性至关重要。该酶能够以良好的选择性拆分不同取代的α-羟基硫酯。设计酶的高分辨率质谱、硼核磁共振光谱和晶体结构分析揭示了该机制的三个步骤（底物结合、氢氧根转移、产物释放）。催化二元组周围一个残基的突变产生了一种酶变体，其催化活性和选择性提高了两倍。
